JP2018198614A - Isolated human lung progenitor cell and use thereof - Google Patents

Abstract

To provide isolated human lung progenitor cells and a use thereof.SOLUTION: Provided are isolated human Nkx 2.1 positive Sox 2 positive proximal airway pluripotent progenitor cells that are proliferative and under selected differentiation conditions differentiate into respiratory tract basal stem cells, ciliated cells, clara cells, neuroendocrine cells, or squamous epithelial cells. Also provided are isolated human Nkx 2.1 positive Sox 9 positive distal pluripotent lung progenitor cells etc. that are proliferative and under selected differentiation conditions differentiate into epithelial lung cells.SELECTED DRAWING: Figure 1

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is filed under 35 USC 119 (e), U.S. Provisional Application No. 61, filed January 13, 2012, the entire contents of each of which is incorporated herein by reference. No. 586,551 and US Provisional Patent Application No. 61 / 619,568 filed Apr. 3, 2012.

The field of the invention relates to isolated human lung progenitor cells, methods of making such isolated human lung progenitor cells, and uses thereof.

Background Lung disease, including respiratory disease, is a major cause of mortality and prevalence worldwide. Current treatments are aimed at reducing the symptoms of pulmonary disease and there are few or no treatments that provide the promise of cure or complete reversal of the disease.

  Transplantation of lung progenitor cells derived from stem cells is one approach that can be used to regenerate lung cells in the body destroyed by injury and disease. There is great interest in the possibility of using stem cells to repair lung epithelium destroyed by injury and disease.

  Stem cells represent a unique population of cells that have the ability to undergo both self-renewal and differentiation. It is beneficial that progenitor cells that can be manipulated are isolated and purified from the subject and then reintroduced into the subject for treatment purposes. By using the subject's own cells, the need to use an associated immunosuppressive treatment is eliminated, thereby maintaining the competency of the subject's immune system. For example, directed differentiation of induced pluripotent stem cells made from a somatic cell sample of interest offers advantages in providing a cell population for self-renewal cell therapy.

Overview Cell differentiation is a complex process that typically occurs through many cell divisions. Partially or fully differentiated cells can be derived from multipotent cells that themselves are derived from multipotent cells, and so on. Each of these multipotent cells can be considered to be a stem cell, but the range of cell types that each can occur can vary considerably. Some differentiated cells also have the ability (eg, reprogramming) to produce cells of greater developmental potential. Such ability can be natural or can be artificially induced by treatment with various factors. In many biological examples, stem cells are also “multipotent” because they can produce progeny of more than one distinct cell type, which means “being stem cells” Not necessary for.

  In addition to the ability to differentiate into a more specific developmental phenotype, self-renewal is another classic part of stem cell definition. In theory, self-renewal can occur by either of two major mechanisms. Stem cells may divide asymmetrically, where one daughter cell retains the stem cell state and the other daughter cells express several distinct other specific functions and phenotypes. Or some of the stem cells in the population can divide symmetrically into two stem cells, thus maintaining some stem cells in the population as a whole, but other cells in the population Produces only differentiated offspring. Formally, cells that start as stem cells can progress towards a differentiated phenotype, but the stem cell phenotype can be "reversed" and re-expressed, which is often "dedifferentiated" by those skilled in the art. "," Reprogramming "or" reverse differentiation ". Such reversal of the differentiation phenotype is generally due to, for example, expressing stem cell specific mRNA or protein (eg, c-Myc, Klf4, Oct4, Sox2 among others) or contacting the cell with a dedifferentiation medium Requires artificial manipulation of cells.

  The methods and compositions provided herein, in part, produce human progenitor cells that are restricted to the lung lineage, and use of such cells to treat lung diseases / disorders or damage to the lungs. About. Whether or not adult stem cells can be isolated from human adult lungs remains controversial in the art, and currently methods for isolating and using adult lung stem cells from humans lack reproducibility. Yes. Thus, the methods and compositions described herein are advantageous over the state of the art in the art and allow the generation of human lung progenitor cells for treatment, tissue engineering, and screening assays To.

  One aspect disclosed herein relates to an enriched population of isolated human Nkx2.1 positive Sox2 positive proximal airway multipotent progenitor cells, or human Nkx2.1 positive Sox2 positive proximal airway multipotent progenitor cells .

  In one embodiment of this aspect, the cell is Tuj1 negative and Pax8 negative.

  In another embodiment of this aspect, the cells are proliferative and differentiate into airway basal stem cells, ciliated cells, Clara cells, neuroendocrine cells, or squamous cells under selected differentiation conditions.

  Similarly, in another aspect, isolated human Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells are provided herein.

  In one embodiment of this aspect, the cell is FoxP2 positive and / or ID2 positive. In another embodiment, the cell is ETV4 / 5 positive.

  In another embodiment of this aspect, the cells are proliferative and differentiate into any epithelial lung cells when placed in selected differentiation conditions.

  In another embodiment of this aspect, the cells, when placed in selected differentiation conditions, are airway basal stem cells, cilia cells, Clara cells, mucin-secreting goblet cells, type I alveolar cells, type II alveolar cells, flattened Differentiates into epithelial cells, bronchoalveolar stem cells, bronchoalveolar junction stem cells, migratory CK14 + cells, or neuroendocrine cells.

  Another aspect described herein relates to an isolated population of isolated human Nkx2.1 positive p63 positive multipotent airway basal stem cells, or isolated human Nkx2.1 positive p63 positive multipotent airway basal stem cells.

  In another embodiment of this aspect, the cells are proliferative and differentiate into ciliated cells, Clara cells, mucin-secreting goblet cells, or basal cells when placed in selected differentiation conditions.

  In another embodiment of this aspect, the multipotent airway basal stem cell does not express a Clara cell marker, a ciliated cell marker, a neuroendocrine cell marker, or a squamous cell marker.

  In another embodiment of this aspect, the cell is Sox2 positive.

  In another embodiment of this aspect, the cell is CK5 positive and / or NGFR positive.

  In another embodiment of this aspect and all other aspects described herein, the cell is a disease-specific cell.

  Another aspect provided herein relates to a composition comprising isolated human Nkx2.1 positive Sox2 positive proximal airway multipotent progenitor cells and a scaffold.

  In another embodiment of this aspect, the scaffold can be implanted in the subject.

  In another embodiment of this aspect, the cell is autologous to the subject into which the composition is to be implanted.

  In another embodiment of this aspect, the scaffold is biodegradable.

  In another embodiment of this aspect, the scaffold comprises natural fibers, synthetic fibers, decellularized lung tissue, or a combination thereof.

  In another embodiment of this aspect, the natural fiber is selected from the group consisting of collagen, fibrin, silk, thrombin, chitosan, chitin, alginic acid, hyaluronic acid, and gelatin.

  In another embodiment of this aspect, the synthetic fibers comprise polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactide-co-glycolide) (PLGA), poly (caprolactone), diol / di Acid-type aliphatic polyester, polyester-amide / polyester-urethane, poly (valerolactone), poly (hydroxylbutyric acid), polybutylene terephthalate (PBT), polyhydroxyhexanoic acid (PHH), polybutylene succinic acid (PBS), and Selected from the group consisting of representative biodegradable aliphatic polyesters such as poly (hydroxyvaleric acid).

  In another embodiment of this aspect, the proximal airway multipotent progenitor cells are Tuj1 negative and / or Pax8 negative.

  Another aspect described herein relates to a composition comprising an isolated human Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cell and a scaffold.

  In one embodiment of this aspect, the multipotent lung progenitor cells are FoxP2 positive and / or ID2 positive. In another embodiment, the cell is ETV4 / 5 positive.

  In another embodiment of this aspect, the scaffold can be implanted in the subject.

  In another embodiment of this aspect, the cell is autologous to the subject into which the composition is to be implanted.

  In another embodiment of this aspect, the scaffold is biodegradable.

  In another embodiment of this aspect, the scaffold comprises natural fibers, synthetic fibers, decellularized lungs, or combinations thereof.

  In another embodiment of this aspect, the natural fiber is selected from the group consisting of collagen, fibrin, silk, thrombin, chitosan, chitin, alginic acid, hyaluronic acid, and gelatin.

  In another embodiment of this aspect, the synthetic fibers comprise polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactide-co-glycolide) (PLGA), poly (caprolactone), diol / di Acid-type aliphatic polyester, polyester-amide / polyester-urethane, poly (valerolactone), poly (hydroxylbutyric acid), polybutylene terephthalate (PBT), polyhydroxyhexanoic acid (PHH), polybutylene succinic acid (PBS), and Selected from the group consisting of representative biodegradable aliphatic polyesters such as poly (hydroxyvaleric acid).

  Another aspect described herein relates to a composition comprising isolated human Nkx2.1 positive p63 positive multipotent airway basal stem cells and a scaffold.

  In one embodiment of this aspect, the airway basal stem cells are CK5 positive and / or NGFR positive.

  In another embodiment of this aspect, the scaffold can be implanted in the subject.

  In another embodiment of this aspect, the cell is autologous to the subject into which the composition is to be implanted.

  In another embodiment of this aspect, the scaffold is biodegradable.

  In another embodiment of this aspect, the scaffold comprises natural fibers, synthetic fibers, decellularized lung tissue, or a combination thereof.

  In another embodiment of this aspect, the natural fiber is selected from the group consisting of collagen, fibrin, silk, thrombin, chitosan, chitin, alginic acid, hyaluronic acid, and gelatin.

  In another embodiment of this aspect, the synthetic fibers comprise polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactide-co-glycolide) (PLGA), poly (caprolactone), diol / di Acid-type aliphatic polyester, polyester-amide / polyester-urethane, poly (valerolactone), poly (hydroxylbutyric acid), polybutylene terephthalate (PBT), polyhydroxyhexanoic acid (PHH), polybutylene succinic acid (PBS), and Selected from the group consisting of representative biodegradable aliphatic polyesters such as poly (hydroxyvaleric acid).

  In another embodiment of this aspect and all other aspects described herein, the cell is a disease-specific cell.

  Similarly, in another aspect, human foregut endoderm cells are differentiated with FGF2, WNT, and BMP4, each to differentiate human foregut endoderm cells into Nkx2.1 positive Tuj1-negative Pax8-negative lung progenitor cells Provided herein is a method of producing human lung progenitor cells or human lung progenitor cell populations that are Nkx2.1 positive, Tuj1 negative, and Pax8 negative, comprising the step of contacting at a sufficient time and concentration.

  In one embodiment of this aspect, the contacting step is performed for at least 2 days.

  In another embodiment of this aspect, the method further comprises Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells, BMP7, FGF7, WNT antagonist, and MAPKK / ERK antagonist, each Nkx2.1 positive Tuj1 negative Pax8 negative Contacting the lung progenitor cells for a period and at a concentration sufficient to differentiate into Nkx2.1 positive Sox2 positive proximal airway multipotent progenitor cells.

  In another embodiment of this aspect, the Wnt antagonist comprises IWR-1.

  In another embodiment of this aspect, the MAPKK / ERK antagonist comprises PD98059.

  In another embodiment of this aspect, the contacting step is performed for at least 4 days.

  In another embodiment of this aspect, the method further comprises Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells with BMP7, FGF7, WNT antagonist and MAPKK / ERK antagonist, each Nkx2.1 positive Tuj1 negative Pax8 negative lung Contacting the progenitor cells for a period and at a concentration sufficient to differentiate into Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells.

  In another embodiment of this aspect, the Wnt antagonist comprises IWR-1.

  In another embodiment of this aspect, the MAPKK / ERK antagonist comprises PD98059.

  In another embodiment of this aspect, the contacting step is performed for at least 4 days.

  In another embodiment of this aspect, foregut endoderm cell culture is derived from embryonic stem (ES) cells or induced pluripotent stem cells (iPSC).

  In another embodiment of this aspect, the method further comprises culturing Nkx2.1 positive Tuj1-negative Pax8-negative cells with B27, BMP7, FGF7, and WNT antagonist, each Nkx2.1-positive Tuj1-negative Pax8-negative lung. Contacting the progenitor cells for a period and at a concentration sufficient to differentiate into Nkx2.1 positive p63 positive multipotent airway basal stem cells.

  In another embodiment of this aspect, the method further comprises contacting Noggin with a culture of Nkx2.1 positive Tuj1 negative Pax8 negative cells.

  In another embodiment of this aspect, the contacting step is performed for at least 10 days.

  Similarly, in another aspect, a subject comprising administering a composition comprising isolated human lung progenitor cells and a pharmaceutically acceptable carrier to a subject having a lung disease or disorder, or lung injury Provided herein are methods for treating lung diseases or disorders in or lung injury.

  In one embodiment of this aspect, the isolated human lung progenitor cells are Nkx2.1 positive Sox2 positive proximal airway multipotent progenitor cells, Nkx2.1 positive Sox9 positive distal multipotent progenitor cells, Nkx2.1 positive It is selected from the group consisting of p63-positive multipotent airway basal stem cells or differentiated cells thereof.

  In another embodiment of this aspect, the proximal airway multipotent progenitor cells are Tuj1 negative and / or Pax8 negative.

  In another embodiment of this aspect, the distal multipotent lung progenitor cells are FoxP2 and / or ID2 positive. In another embodiment, the cell is ETV4 / 5 positive.

  In another embodiment of this aspect, the airway basal stem cells are CK5 positive and / or NGFR positive.

  In another embodiment of this aspect, the composition is administered to the lung.

  In another embodiment of this aspect, the isolated human lung progenitor cells are autologous to the subject to which the composition is administered.

  In another embodiment of this aspect, the composition further comprises a scaffold.

  In another embodiment of this aspect, the scaffold can be implanted in the subject.

  In another embodiment of this aspect, the scaffold is biodegradable.

  In another embodiment of this aspect, the scaffold comprises natural fibers, synthetic fibers, decellularized lung tissue, or a combination thereof.

  In another embodiment of this aspect, the scaffold comprises a substance that promotes the differentiation of isolated human lung progenitor cells.

  In another embodiment of this aspect, the composition is formulated for aerosol delivery.

  Another aspect provided herein is produced by in vitro differentiation of human disease-specific lung progenitor cells in the presence and absence of (a) a candidate substance for treating a lung disease or disorder. Culturing a human disease-specific airway cell population; (b) comparing the expression or activity of at least one marker up-regulated in the disease in the presence and absence of the candidate substance, or down in the disease If the expression or activity of at least one up-regulated disease marker is reduced in the step of comparing the expression or activity of at least one regulated marker, or the expression of at least one down-regulated disease marker Or if the activity increases, candidates for treating lung disease or disorder in a subject And a method for screening a substance for treating a pulmonary disease or disorder, comprising the step of identifying a candidate substance.

  In one embodiment of this aspect, the method further comprises the step of differentiating the population of isolated human disease-specific lung progenitor cells into a culture of human disease-specific airway cells prior to step (a).

  In another embodiment of this aspect, the method further comprises, prior to step (a), an induced pluripotent stem cell population derived from a subject having a lung disease or disorder, isolated human disease-specific lung progenitor cells Including differentiating into a population.

  In another embodiment of this aspect, the candidate substance comprises a small molecule, protein, polypeptide, antibody or antigen-binding fragment thereof, or nucleic acid.

  In another embodiment of this aspect, the human lung progenitor cells are human Nkx2.1 positive Sox2 positive proximal airway multipotent progenitor cells, human Nkx2.1 positive Sox9 positive distal multipotent progenitor cells, and human Nkx2 Selected from the group consisting of .1 positive p63 positive multipotent airway basal stem cells.

  In another embodiment of this aspect, human Nkx2.1 positive Sox2 positive proximal airway multipotent progenitor cells and human Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells are Nkx2.1 positive Tuj1 negative Pax8 negative Lung progenitor cells, BMP7, FGF7, WNT antagonist, and MAPKK / ERK antagonist, Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells, Nkx2.1 positive Sox9 positive proximal airway multipotent progenitor cells or Nkx2 Produced by a method comprising contacting at a sufficient time and concentration to differentiate into .1 positive Sox9 positive distal multipotent lung progenitor cells.

  In another embodiment of this aspect, the contacting step is performed for at least 4 days.

  In another embodiment of this aspect, human Nkx2.1 positive p63 positive multipotent airway basal stem cells are Nkx2.1 positive Tuj1 negative Pax8 negative cells, B27, BMP7, FGF7, and WNT antagonist, each Nkx2. It is produced by a method comprising contacting 1-positive Tuj1-negative Pax8-negative lung progenitor cells with a period and concentration sufficient to differentiate into Nkx2.1-positive p63-positive multipotent airway basal stem cells.

  In another embodiment of this aspect, the method further comprises contacting Noggin with Nkx2.1 positive Tuj1 negative Pax8 negative cells.

  In another embodiment of this aspect, the contacting step is performed for at least 10 days.

  Another aspect provided herein includes (a) culturing Nkx2.1 positive Tuj1 negative Pax8 negative human lung progenitor cells in the presence and absence of candidate differentiation substances, (b) Compare the expression or activity of at least one marker that is up-regulated upon differentiation of lung progenitor cells to a more differentiated state in the presence and absence, or to the more differentiated state of lung progenitor cells Comparing the expression or activity of at least one marker that is down-regulated upon differentiation, if the expression or activity of at least one up-regulated differentiation marker decreases or is at least one down-regulated Use the candidate substance to induce differentiation of isolated human lung progenitor cells in the subject Comprising it can be shown, to a method of screening a substance that induces differentiation of human lung progenitor cells.

  In one embodiment of this aspect, the method further comprises differentiating embryonic stem cells or induced pluripotent stem cells into human lung progenitor cells prior to step (a).

  In another embodiment of this aspect, the candidate substance comprises a small molecule, protein, polypeptide, antibody, or nucleic acid.

  In another embodiment of this aspect, the human lung progenitor cells are human Nkx2.1 positive Sox2 positive proximal airway multipotent progenitor cells, human Nkx2.1 positive Sox9 positive distal multipotent progenitor cells, and human Nkx2 Selected from the group consisting of .1 positive p63 positive multipotent airway basal stem cells.

  In another embodiment of this aspect, human Nkx2.1 positive Sox2 positive proximal airway multipotent progenitor cells and human Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells are Nkx2.1 positive Tuj1 negative Pax8 negative Human lung progenitor cells, BMP7, FGF7, WNT antagonist, and MAPKK / ERK antagonist, Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells, Nkx2.1 positive Sox9 positive proximal airway multipotent progenitor cells or Produced by a method comprising contacting at a sufficient time and concentration to differentiate into Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells.

  In another embodiment of this aspect, the contacting step is performed for at least 4 days.

  In another embodiment of this aspect, human Nkx2.1 positive p63 positive multipotent airway basal stem cells are Nkx2.1 positive Tuj1 negative Pax8 negative cells, B27, BMP7, FGF7, and WNT antagonist, each Nkx2. It is produced by a method comprising contacting 1-positive Tuj1-negative Pax8-negative lung progenitor cells with a period and concentration sufficient to differentiate into Nkx2.1-positive p63-positive multipotent airway basal stem cells.

  In another embodiment of this aspect, the method further comprises contacting Noggin with Nkx2.1 positive Tuj1 negative Pax8 negative cells.

  In another embodiment of this aspect, the contacting step is performed for at least 10 days.

  Another aspect provided herein includes a lung comprising the cell of claim 1, 5, or 9, a pharmaceutically acceptable carrier, and instructions for treating a lung disease or disorder. It relates to a kit for treating a disease or a pulmonary disorder.

  In one embodiment of this aspect, the kit further comprises a scaffold.

  Similarly, in another aspect, a candidate substance is screened comprising the cell of claim 1, 5, or 8, one or more substances for detecting lung-specific cell surface markers, and instructions thereof Kits for doing so are provided herein.

  In one embodiment of this aspect, the kit further comprises a cell culture medium, a growth factor, and / or a differentiation agent.

  Another aspect provided herein is (i) two or more of BMP4, FGF2, WNT, BMP7, FGF7, WNT antagonist, Noggin, B27, and retinoic acid, optionally provided in unit dose Many; (ii) optionally cell culture medium; (iii) one or more substances for detecting lung cell specific surface markers; and (iii) human stem cells to human lung progenitor cells, including instructions thereof And a kit for differentiation.

  Similarly, provided herein is a cell or tissue culture medium for producing definitive endoderm from iPSCs or ESCs comprising B27, activin A, and ZSTK474. In one embodiment, the medium comprises B27 at a concentration of 1% to 5% (eg, 2%). In another embodiment, the medium comprises activin A at a concentration of 10-40 ng / mL (eg, 20 ng / mL). In another embodiment, the medium contains ZSTK474 at a concentration of 0.2-0.5 μM. In another embodiment, the basal medium comprises components of RPMI medium.

  Similarly, provided herein is a cell or tissue culture medium for making Nkx2.1 positive lung progenitor cells comprising CHIR9902, PIK-75, dorsomorphin, and FGF2. In some embodiments, the medium comprises CHIR9902 at a concentration of 0.1-1 μM. In another embodiment, the medium contains PIK-75 at a concentration of 0.01-0.1 μM. In another embodiment, the medium comprises dorsomorphin at a concentration of 1-5 μM. In another embodiment, the medium comprises FGF2 at a concentration of 10-100 ng / mL. In another embodiment, the medium further comprises the group consisting of GF-109203X, Ro31-8220, Pp242, PIK-75, ZSTK474, PMA, carvedilol, corticosterone, triclabendazole, benproperin phosphate, phenothiazine, and methotrexate. Contains the drug of choice.

  Similarly, herein, human foregut endoderm cells are treated with Wnt agonists, PIK3 kinase inhibitors, BMP antagonists, and GF-109203X, Ro31-8220, Pp242, PIK-75, ZSTK474, PMA, carvedilol, corticosterol. For the differentiation of human foregut endoderm cells into Nkx2.1-positive Tuj1-negative Pax8-negative lung progenitor cells, each selected from the group consisting of N, triclabendazole, benproperine phosphate, phenothiazine, and methotrexate Methods are provided for producing human lung progenitor cells or human lung progenitor cell populations that are Nkx2.1 positive, Tuj1 negative, and Pax8 negative, comprising contacting for a sufficient period and concentration.

  In one embodiment, the contacting step is performed for at least 2 days.

  In another embodiment, the Wnt agonist comprises CHIR9902. In another embodiment, the concentration of CHIR9902 is in the range of 0.1-1 μM.

  In another embodiment, the PI3 kinase inhibitor comprises PIK-75. In another embodiment, the concentration of PIK-75 comprises 0.01-0.1 μM.

  In another embodiment, the BMP antagonist comprises dorsomorphin. In another embodiment, the concentration of dorsomorphin comprises 1-5 μM.

  In another embodiment, the growth factor comprises FGF2. In another embodiment, the concentration of FGF2 comprises 10-100 ng / mL.

  Similarly, in another aspect, the method comprises contacting iPSC or ESC with B27, activin A, and ZSTK474 for a period and at a concentration sufficient to each differentiate iPSC or ESC into definitive endoderm cells. A method for producing a definitive endoderm cell or definitive endoderm cell population is provided.

  In one embodiment, the production of definitive endoderm cells is determined by FACS analysis with FOXA2 / SOX17 co-staining or a combination of cKit / CXCR4 and / or cKit / EpCAM.

  In another embodiment, the concentration of B27 comprises 1% to 5% (eg, 2%).

  In another embodiment, the concentration of activin A is 10-40 ng / mL (eg, 20 ng / mL).

  In another embodiment, the concentration of ZSTK474 is 0.2-0.5 μM.

FIG. 5 shows the overall strategy for staged differentiation of airway progenitor cells from mouse ESCs. The figure shows an approach for developing an efficient and reproducible protocol for producing embryonic airway epithelial progenitor cells from mouse ESCs. Signaling network is responsible for ESC differentiation into definitive endoderm, anteriorization of endoderm to foregut endoderm, induction of early lung endoderm from foregut endoderm, and generation of embryonic airway progenitor cells Facilitate. Data showing that forward migration of endoderm to foregut endoderm promotes Nkx2.1 + cell differentiation. FIG. 2A shows that administration of 20 ng / ml BMP4, 20 ng / ml FGF2, and 5 nM GSK3iXV to definitive endoderm resulted in hindgut differentiation with CDX2 expression and minimal NKX2.1 induction. The scale bar is 50 μm. FIG. 2B shows 2-day treatment of definitive endoderm with different combinations of the BMP4 / TGFβ agonists and antagonists listed in the table. Foxa2 + / Sox2 + cells were quantified as a percentage of positive cells in the total number of Foxa2 + cells. To induce NKX2.1 expression, cells were further treated with 20 ng / ml BMP4, 20 ng / ml FGF2, and 5 nM GSK3iXV. The percentage of Nkx2.1 + cells was quantified as the percentage of total cells present. All data were averaged from 3 independent experiments. FIG. 5 shows that BMP4, FGF2, and WNT signaling are required for lung specification from foregut endoderm cells. FIG. 3A shows a strategy and approximate time for generating Nkx2.1 + cells from foregut endoderm cells. FIG. 3B is a list of various combinations of BMP4, FGF2, WNT and their antagonists to induce Nkx2.1 +. FIG. 3C shows the percentage of NKX2.1 recorded by Nkx2.1 + cell count in total cell count, with an average of 3 independent experiments. We show that Smad-dependent BMP signaling is required for lung differentiation. NKX2.1 expression with 20 ng / ml FGF2 and 5 nM GSK3iXV alone (control) or with additional BMP4 (20 ng / ml), BMP2 (20 ng / ml), BMP7 (20 ng / ml), BMP4 ( 20 ng / ml) + 10 μM Dorsomorphin or BMP4 (20 ng / ml) + 1 μM PD98059 (data not shown). FIG. 4 shows that BMP signaling through SMAD-dependent pathways is required for lung Nkx2.1 + differentiation. In contrast, signaling through the MAP kinase pathway is not required for Nkx2.1 expression. Dorsomorphin inhibits BMP signaling through the SMAD pathway, while PD98059 inhibits MAPK signaling. FIG. 6 shows the production of embryonic airway progenitor cells from multipotent lung endoderm cells. FIG. 5A shows a strategy and time schedule for generating embryonic airway progenitor cells from multipotent lung endoderm cells. FIG. 5B is a summary of the different signaling switches in the proximal airway and distal lung bud tip during the pseudoglandular phase of lung development. Treatment of Nkx2.1 + endoderm cells with D9 with RA-added B27, 20 ng / ml BMP7, 20 ng / ml FGF7, 100 nM IWR-1 (WNT antagonist), and 100 ng / ml Noggin for 2 days After that, immunofluorescent staining of NKX2.1 and SOX2 was performed (data not shown). In addition, Nkx2.1 + lung endoderm cells were cultured in D9 with RA supplemented B27, 20 ng / ml BMP7, 20 ng / ml FGF7, 100 nM IWR-1 (WNT antagonist), and 1 μM PD98059 for 2 days After treatment, immunofluorescent staining of NKX2.1 and SOX2 was performed. The scale bar is 50 μm (data not shown). Data from immunofluorescence staining showed that there was a subpopulation of Nkx2.1 + cells that were positive for p63 (data not shown). FIG. 2 shows an exemplary strategy and time schedule for generating Nkx2.1 + lung multipotent progenitor cells from human iPSCs. The data from the experiment shows the staged differentiation of Nkx2.1 + lung progenitor cells from human iPSC. After treatment for 4 days in the presence of 2% B27 supplement, activin A (100 ng / ml) and 5 μM PI3 kinase inhibitor LY294002 in RPMI-1640 medium, definitive endoderm was obtained in high yield from CF1 RiPS cells, More than 90% of the cells co-expressed the transcription factors SOX17 and FOXA2 (data not shown). Pre-endoderm by detecting SOX2 expression in Foxa2 + cells derived from CF1 RiPS cells after 4 days of treatment with 500 nM A-83-01 (TGFβ antagonist) and 100 ng / ml Noggin (BMP4 antagonist) Forward migration to intestinal endoderm cells was observed (data not shown). With D8, NKX2.1 staining was performed after forward transfer to foregut cells for 4 days in serum-free medium containing 20 ng / ml BMP4, 20 ng / ml FGF2, and 5 nM GSK3iXV (data not shown) . Immunofluorescence shows that Nkx2.1 positive cells are co-stained with SOX2, SOX9, TUJ1, and PAX8, thereby eliminating the presence of thyroid and neuronal differentiation and multipotent distal terminal progenitor cells and Nkx2.1 + / Sox2 + airway progenitor cells were demonstrated (data not shown). In addition, confocal images after immunofluorescence staining show that some Nkx2.1 + spheres contain basal cells positive for p63 (data not shown). FIG. 6 shows a treatment strategy and approximate time for producing definitive endoderm. Immunofluorescence of FOXA2 and SOX17 on day 5 indicates that> 90% of the cells were definitive endoderm made from p2A mESC and V6.5 mESC (data not shown) . FIG. 2 is a table and figure summarizing immunofluorescence data for specific combinations of markers that separate Nkx2.1 + brain, thyroid, and lung organ discs in mouse embryos. Immunofluorescence imaging of Nkx2.1 and SOX2 was performed in ventral forebrain, thyroid, and lung from E8.75 to E9 (data not shown). Immunofluorescence imaging was also performed for Nkx2.1, Tuj1, and Sox9 in the ventral forebrain from E8.75 to E9 and for Nkx2.1, Pax8, and Sox9 in the thyroid. It is a graph which shows the gene expression analysis of the fate of ESC induction | guidance | derivation anterior endoderm. Nkx2.1, Sox2, FoxN1, Pax9, Tbx1, Pax8 in mouse ESC or human iPSC, corresponding to AE after treatment with definitive endoderm (DE), anterior endoderm (AE) and Nkx2.1 induced growth factor cocktail Quantitative expression of Sox9 and FoxP2 mRNA. n = 3 biological replicates, expression levels are normalized to ESC or iPSC levels. FIG. 5 is a photomicrograph showing the presence of functional cilia on the surface of airway cells differentiated from human lung progenitor cells described herein. The cilia are observed to wave in concert, indicating that the airway cells are functional. It is a figure which shows the possibility of differentiation regarding Nkx2.1 + Tuj1-Pax8- cell. Without wishing to be bound by theory, FIG. 11 shows two possible mechanisms by which downstream human lung progenitor cells can differentiate from Nkx2,1 + Tuj1-Pax8− cells. FIG. 2 is a schematic diagram showing an exemplary stepwise airway progenitor cell differentiation protocol from iPSC to airway progenitor cells. It is a schematic diagram showing a variation of a protocol for producing definitive endoderm from human iPSC and ESC. The protocol allows for the production of definitive endoderm with high efficiency and consistency. Figure 5 shows unbiased chemical screening to enhance lung differentiation from anterior endoderm. FIG. 14A shows a schematic diagram showing a chemical screening platform. FIG. 14B is a table showing lead compounds that promoted production of Nkx2.1 + lung progenitor cells.

DETAILED DESCRIPTION The compositions and methods described herein have discovered, in part, a method for producing isolated human lung progenitor cells from embryonic stem (ES) cells or induced pluripotent cells (iPSCs). About. The presence and isolation of adult progenitor cells from lung tissue is controversial and has only limited success, and therefore the methods and compositions described herein are isolated humans. Lung progenitor cells have the advantage that they can be produced in quantities useful for screening assays or treatment of lung diseases / disorders or lung damage. Furthermore, the cell compositions provided herein have not been previously isolated and / or cultured from human lung tissue.

Definitions As used herein, the term “human stem cell” means a human cell that can self-renew and differentiate into at least one cell type. The term “human stem cells” includes human stem cell lines, human-derived iPS cells, human embryonic stem cells, human pluripotent cells, human multipotent stem cells, or human adult stem cells.

  The term “multipotency” as used herein means that a cell can differentiate into multiple different phenotypes. Multipotent cells are generally capable of differentiating into only one germ line lineage cell. This is in contrast to pluripotent cells, which by definition can differentiate into cells of all three germ layers. Pluripotent cells are mainly characterized by their ability to differentiate into all three germ layers using, for example, a nude mouse teratoma formation assay. Although pluripotency is also evidenced by the expression of embryonic stem (ES) cell markers, a preferred test for pluripotency is the demonstration of the ability of the three germ layers to differentiate into cells. Pluripotent cells typically have the ability to divide in vitro for long periods of time, eg, longer than 1 year or more than 30 passages.

  The term “human lung progenitor cells” as used herein is a general term that means any progenitor cell that is restricted to the lung lineage but also retains the ability to self-renew. One of the first stages of restraint to the lung lineage is the emergence of the stem cell marker Nkx2.1, which can also be detected in cells of the thyroid and brain lineage. Therefore, for the purpose of this explanation, the first progenitor cells that are restricted to the lung lineage and are encompassed by the term “human lung progenitor cells” express Nkx2.1 but lack the Tuj1 and Pax8 cell surface markers Cell. Any progenitor cell that can differentiate from Nkx2.1 positive Tuj1 negative Pax8 negative cells and retains self-renewal ability is encompassed by the term “human lung progenitor cells”. Other examples of human lung progenitor cells described herein include Nkx2.1 positive Sox2 positive proximal multipotent airway progenitor cells, Nkx2.1 positive Sox9 positive distal multipotent embryonic progenitor cells, and Nkx2 Examples include, but are not limited to, .1 positive p63 positive basal airway stem cells. In some embodiments, human lung progenitor cells, when placed in selected differentiation conditions, are airway basal stem cells, ciliated cells, Clara cells, mucin-secreting goblet cells, type I alveolar cells, type II alveolar cells, or nerves Differentiate into endocrine cells. Human lung progenitor cells are not tumor cells or cancer cells. In one aspect, human lung progenitor cells are not derived from embryos or embryonic stem cells or from other cells derived in embryo culture. In some embodiments, human lung progenitor cells differentiate from autologous or non-self cells. In one embodiment, the human lung progenitor cells are not genetically modified cells or are not derived from genetically modified cells.

  As used herein, the term “distal multipotent lung progenitor cells” includes airway basal stem cells, cilia cells, Clara cells, neuroendocrine cells, squamous cells, type I alveolar cells, type II alveolar cells, Bronchoalveolar stem cells (BASC), lung progenitor cells present at the bronchoalveolar junction (BADJ) in the terminal bronchiole, type I and type II alveolar cell progenitors, and / or lung damage (e.g. Nkx2. Capable of differentiating into all types of epithelial cells of the lung, including but not limited to migratory CK14 + cells for both airway epithelial cells and alveolar cells responding to influenza infection) It means 1 positive Sox9 positive cell.

  As used herein, the term “proximal airway multipotent progenitor cell” is differentiated into airway basal stem cells, cilia cells, Clara cells, neuroendocrine cells, squamous cells, or alveolar cells after lung injury Therefore, it means Nkx2.1-positive Sox2-positive cells that can differentiate into migratory CK14 + cells that migrate to the bronchoalveolar position.

  The term “differentiated cell” means any primary cultured cell whose term is not pluripotent in its native form as defined herein. Those skilled in the art recognize that there is a spectrum of differentiation from totipotent or pluripotent cells at one end to fully differentiated cells that do not have the normal ability to naturally differentiate into any other phenotype . Thus, pluripotent cells are differentiated compared to totipotent cells, and multipotent cells are differentiated compared to pluripotent cells. In some embodiments, the term “differentiated cells” is also derived from cells of a less specific cell type (eg, undifferentiated cells or reprogrammed cells) in which the cells are undergoing a cell differentiation process. Means a cell of a more specific cell type.

  The term “positive” (eg, Nkx2.1 positive) as used herein when referring to cells that are positive for a marker is a flow such as immunofluorescence microscopy or fluorescence activated cell sorting (FACS). Using cytometry means that cell surface markers can be detected in cells above background levels. Alternatively, the term “positive” or “express a marker” means that the expression of mRNA encoding a cell surface marker or an intracellular marker can be detected above background levels using RT-PCR. Means. Expression levels of cell surface markers or intracellular markers were obtained from negative controls (ie, cells known to lack the marker) or isotype controls (ie, without appropriate specificity but cell protein, lipid Or a control antibody that only performs non-specific binding to carbohydrate). Thus, cells that "express" a marker (or "positive for a marker") have a detectable expression level that exceeds the expression level determined for a negative control for that marker.

  As used herein, the term “negative” (or the term “not expressed”) when referring to cells that are negative with respect to a marker is an immunofluorescence microscope or fluorescence activated cell sorting (FACS), etc. Using flow cytometry means that cell surface markers cannot be detected on cells above background levels. Alternatively, the terms “negative” or “not expressed” mean that the expression of intracellular marker or cell surface marker mRNA cannot be detected above background levels using RT-PCR. Expression levels of cell surface markers or intracellular markers are derived from negative controls (ie cells known to lack the marker) or isotype controls (ie cell proteins, lipids that do not have the appropriate specificity) Or a control antibody that only performs non-specific binding to carbohydrate). For this reason, cells that do not "express" a marker appear to be similar to the negative control for that marker.

  As used herein, the phrase “cells are proliferating” refers to the ability of stem cells to self-renew. Self-renewal can occur by either of two major mechanisms. Stem cells may divide asymmetrically, where one daughter cell retains the stem cell state and the other daughter cells express several distinct other specific functions and phenotypes. Or, some of the stem cells in the population can divide symmetrically into two stem cells, thus maintaining several stem cells in the population as a whole, while others in the population Cells produce only differentiated progeny.

  As used herein, the term “differentiating ability” means the ability of a stem cell, progenitor cell, pluripotent cell, or multipotent cell to differentiate into a more differentiated subset of cells. The term “differentiating ability” does not encompass moving backwards along the differentiation spectrum so that cells are produced that have a greater differentiation ability than the parent cell. That is, the term “differentiating ability” does not encompass reprogramming methods that shift cells to a less differentiated state.

  In the context of cell ontogeny, the term “differentiate” or “differentiated” indicates that a “differentiated cell” is a cell that has progressed further into a developmental pathway downstream of its progenitor cells. It is a relative term. Thus, in some embodiments, reprogrammed cells, the term of which is defined herein, can differentiate into lineage-restricted progenitor cells (such as human lung progenitor cells), which then Can further differentiate into other types of progenitor cells in the downstream pathway (such as tissue-specific progenitor cells such as proximal airway multipotent progenitor cells) and then play a characteristic role in certain tissue types, Differentiated into terminally differentiated cells that may or may not retain additional proliferative capacity.

  As used herein, the term “dedifferentiation” or “reprogramming” or “reverse differentiation” creates a cell that reexpresses more stem cell phenotypes or fewer differentiation phenotypes than the cells from which it is derived. Means the process to do. For example, multipotent cells can be dedifferentiated into pluripotent cells. That is, dedifferentiation shifts cells in the opposite direction along the differentiation spectrum from totipotent cells to fully differentiated cells. Typically, reversal of the differentiation phenotype of a cell requires artificial manipulation of the cell, for example by expressing stem cell specific mRNA and / or protein. Reprogramming is typically not observed under natural conditions in vivo or in vitro.

  As used herein, the term “somatic cell” refers to any cell other than a germ cell, a cell present in or obtained from a preimplantation embryo, or a cell resulting from the growth of such a cell in vitro. Means. In other words, somatic cell means any cell that forms the body of an organism, as opposed to germline cells. All cell types present in the mammalian body, except sperm and ovum, the cells from which sperm and ova are made (genital cells), and undifferentiated stem cells are somatic cells, internal organs, skin, bones, Blood and connective tissue are all composed essentially of somatic cells. In some embodiments, a somatic cell is a “non-embryonic somatic cell” that means a somatic cell that is not present in or obtained from the embryo and that is not due to the growth of such a cell in vitro. In some embodiments, a somatic cell refers to a cell that is present in or obtained from an organism other than an embryo or fetus, or that results from in vitro growth of such a cell. Somatic cells. Except as otherwise noted, methods for reprogramming differentiated cells (eg, methods for generating iPSCs) can be performed both in vivo and in vitro (in vivo, differentiated cells are not targeted). In vitro is performed using isolated differentiated cells maintained in culture).

  As used herein, the term “adult cell” means a cell found throughout the body after embryo development.

  An “isolated cell” as used herein refers to a cell that has been removed from an organism that was originally found in such a cell or is a descendant of such a cell. Optionally, the cells are cultured in vitro, for example in the presence of other cells. Optionally, the cell is later introduced into a second organism or reintroduced into the organism from which the cell (or a cell that is a descendant of the cell) was isolated.

  As used herein, the term “isolated population” with respect to an isolated cell population means a cell population that has been removed and separated from a mixed or heterogeneous cell population. In some embodiments, an isolated population is a substantially pure cell population as compared to a heterogeneous population from which cells are isolated or enriched. In some embodiments, the isolated population is an isolated human lung progenitor cell population, eg, compared to a heterogeneous cell population comprising human lung progenitor cells and cells from which human lung progenitor cells are derived, A substantially pure human lung progenitor cell population.

  The term “substantially pure” refers to a particular cell population, at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably with respect to the cells that make up the total cell population. By cell population that is at least about 95% pure. That is, with respect to a lung progenitor cell population, the terms “substantially pure” or “essentially purified” refer to cells that are not lung progenitor cells as defined herein by more than about 20%. Refers to a cell population containing less, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% To do.

  The terms “enrich” or “enriched” are used interchangeably herein and refer to one type of cell, such as human lung progenitor cell compositions and cells used in the methods described herein. Yield of at least 10%, at least 15%, at least 20%, at least 25%, at least 30 relative to the fraction of cells of that type in the starting biological sample, culture, or preparation %, At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%.

  As used herein, “proliferate” and “proliferation” refer to an increase in cell number (growth) in a population by cell division. Cell proliferation is generally understood to result from coordinated activation of a number of signaling pathways in response to the environment, including growth factors and other mitogens. Cell proliferation can also be promoted by the action of intracellular or extracellular signals and release from mechanisms that block or negatively affect cell proliferation.

  The terms “regeneration” or “self-renewal” or “proliferation” are used interchangeably herein and refer to the process of a cell that makes more copies (eg, replicas) of the cell itself. In some embodiments, pulmonary progenitor cells measure by the presence or absence of one or more cell surface markers by dividing into long term and / or months to years into the same undifferentiated cells (eg, Can play itself). In some examples, proliferation means an increase in the number of lung progenitor cells due to repeated division of one cell into two identical daughter cells.

  As used herein, the term “separation” or “selection” refers to isolating different cell types into one or more populations, and targeting an isolated population that is enriched in a specific target stem cell population Means collecting as a cell population. Selection should be done using a positive selection whereby the target enriched cell population is retained, or a negative selection where non-target cell types are discarded (thus enriching the desired target cell type in the remaining cell population) Can do.

  As used herein, the term “positive selection” means the selection of a desired cell type by retaining the cells of interest. In some embodiments, positive selection involves the use of a substance to help retain the cells of interest, such as an antibody with a specific binding affinity for a desired or surface antigen on the target cell, such as an antibody. With use. In some embodiments, positive selection is performed in the absence of a positive selection agent, eg, positive selection of a target cell type is based on either the cell size, density and / or morphology of the target cell type, eg, “touch free”. Or can occur in a closed system.

  The term “negative selection” as used herein selects unwanted or non-target cells for depletion or disposal, thereby retaining (and thus enriching) the desired target cell type. Means that. In some embodiments, the negative selection is the use of substances that aid in the selection of unwanted cells for disposal, such as monoclonal antibodies having specific binding affinity for surface antigens on unwanted or non-target cells, etc. With the use of negative selection substances. In some embodiments, negative selection does not involve a negative selection agent. In some embodiments, negative selection is performed in the absence of a negative selection agent, eg, negative selection of unwanted (non-target) cell types to be discarded, cell size, density of undesirable (non-target) cell types. And / or may occur in a “touch free” or closed system based on any of the forms.

  As used herein, the term “marker” is used to describe the characteristics and / or phenotype of a cell. The marker can be used to select cells that contain the feature of interest and can vary depending on the specific cell. A marker is a feature whether or not it is a morphological, functional, or biochemical (enzymatic) feature of a cell of a particular cell type or a molecule expressed by the cell type. In one aspect, such a marker is a protein. Such proteins may carry epitopes of antibodies or other binding molecules available in the art. However, a marker can consist of any molecule found in a cell, including but not limited to proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological features or traits include, but are not limited to, shape, size, nuclear to cytoplasm ratio. Examples of functional characteristics or traits include, but are not limited to, the ability to adhere to a particular substrate, the ability to incorporate or exclude a particular dye, the ability to migrate under certain conditions, and the ability to differentiate along a particular lineage. It is not done. The marker can be detected by any method available to those skilled in the art. The marker can also be absent of morphological features or absence of proteins, lipids, etc. A marker can be a combination of unique features for the presence or absence of polypeptides, and a panel of other morphological features. In one embodiment, the marker is a cell surface marker. Exemplary cell surface markers expressed on lung progenitor cells include Sox2, Sox9, p63, FoxP2, ETV4 / 5, FoxA2, Nkx2.1, Gata6, ID2, CK5, NGFR, FoxJl, CCSP, Scgb3a2, Muc5ac , T1a, Spc, and Scgn, but are not limited to these. In some embodiments, the absence of cell surface markers can be used to distinguish lung progenitor cells from cells of another lineage (eg, thyroid or brain lineage). Exemplary cell surface markers that are not present in lung progenitor cells or differentiated lung cells include, but are not limited to, Tuj1 and Pax8. One skilled in the art will recognize that cell surface markers may be present at specific points in development or on specific lung progenitor cell types. For example, Sox2 is expressed in progenitor cells of the anterior endoderm but not in more differentiated lung progenitor cells, such as distal multipotent lung progenitor cells, and as progenitor cell differentiation proceeds, airway progenitors Reactivated in cells such as cells. In this way, cell surface markers can be used in combination with a positive selection strategy for one lung progenitor cell, as well as for other lung progenitor cells, depending on the specific differentiation stage of the desired lung progenitor cell selected. Can be used in combination with a negative selection strategy.

  As used herein, the term “scaffold” means a structure comprising a biocompatible material that provides a suitable surface for cell adhesion and growth. The scaffold can further provide mechanical stability and support. The scaffold may be present in a particular shape or type to affect or delimit the three-dimensional shape or type that the growing cell population takes. Such shapes or molds include thin films (eg, molds having two dimensions substantially larger than the third dimension), ribbons, strings, sheets, flat disks, cylinders, spheres, three-dimensional amorphous shapes, etc. Including, but not limited to.

  As used herein, the term “implantable in a subject” means any non-viable (eg, cell-free) implantable structure that does not produce a recognizable immune response in a host organism upon implantation. Thus, the implantable structure must not be a stimulant, for example, or contain a stimulant, or contain LPS or the like.

  As used herein, the term “biodegradable” refers to the ability of a scaffold to degrade under physiological conditions, for example, conditions that do not deleteriously affect the cell viability of delivered cells or cells in vivo. means. Such a biodegradable scaffold is preferably not or does not contain a stimulant or allergen that can cause a systemic response in a subject in which the composition is implanted. In some embodiments, biodegradable means that the scaffold can be metabolized and the metabolite can disappear from the subject by physiological excretion mechanisms (eg, urine, stool, liver detoxification, etc.).

  The term “treating” as used herein includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. For example, the terms “treat” and “treatment” refer to the effectiveness of the composition so that the subject has a reduction in at least one symptom of disease or an improvement in disease, eg, has a beneficial or desired clinical outcome. Meaning that an amount, eg, an effective amount of a composition comprising a lung progenitor cell population, is administered to a subject. For the purposes of this disclosure, beneficial or desired clinical outcomes, whether detectable or undetectable, can reduce one or more symptoms, reduce the extent of the disease, stabilize the disease (e.g., , Not aggravated), delay or delay of disease progression, improvement or alleviation of disease state, and remission (partial or complete). In some embodiments, treating may mean prolonging survival as compared to expected survival if not receiving treatment. Thus, those skilled in the art will recognize that treatment can ameliorate the disease state but is not a complete cure for the disease. In some embodiments, treatment can include prevention. However, in an alternative embodiment, treatment does not include prevention.

  “Treatment” of a lung disorder, lung disease, or lung injury (eg, acute lung injury) as referred to herein refers to a therapeutic intervention that stabilizes or improves lung or airway function. That is, "treatment" is directed to respiratory organ function. Lung or airway function is at least 10% compared to such function prior to such treatment, and preferably at least 20%, 30%, 40%, 50%, 75%, 90%, 100% or A therapeutic approach that stabilizes or improves beyond that, eg, 2x, 5x, 10x or more to full function and including full function is considered an effective treatment. An effective treatment need not cure or directly affect the underlying cause of a lung disease or disorder that is considered to be an effective treatment.

  The phrase “pharmaceutically acceptable” is valid within the scope of sound medical judgment herein without causing excessive toxicity, irritation, allergic reactions, or other problems or complications. Used to mean a compound, material, composition, and / or dosage form suitable for use in contact with human and animal tissues, commensurate with the benefit / risk ratio.

  As used herein, the terms “pharmaceutically acceptable”, “physiologically acceptable” and grammatical variations thereof used when referring to compositions, carriers, diluents, and reagents are: Used interchangeably to indicate that the material can be administered to a mammal without causing undesirable physiological effects such as nausea, dizziness, upset stomach, and others. A pharmaceutically acceptable carrier does not promote the generation of an immune response to the substance with which it is mixed, unless it is desirable to do so. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable preparations, either as liquid solutions or suspensions, but are solid agents suitable for making solutions or suspensions in liquid prior to use Shapes can be prepared as well. The preparation can also be emulsified or presented as a liposomal composition. The active ingredient can be admixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or others and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient. The therapeutic composition of the present invention may include pharmaceutically acceptable salts of the components. Pharmaceutically acceptable salts are acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid and others (formed by the free amino group of the polypeptide. Included). Salts formed by free carboxyl groups also include inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, as well as isopropylamine, trimethylamine, 2-ethylamino It can be derived from organic bases such as ethanol, histidine, procaine, and others. Physiologically acceptable carriers are well known in the art. Exemplary liquid carriers include no ingredients other than the active ingredient and water, or buffers such as both solutions such as sodium phosphate, physiological saline, or phosphate buffered saline at physiological pH values. A sterile aqueous solution containing an agent. Still further, the aqueous carrier may contain more than one buffer salt and salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. The liquid composition may also include a liquid phase in addition to and excluding water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active ingredient used in conjunction with the methods described herein that are effective in the treatment of a particular disorder or condition can be determined by standard clinical techniques, depending on the nature of the disorder or condition.

  As used herein, “prevention” or “preventing” when used in connection with a disease, disorder, or symptom thereof, refers to a reduction in the likelihood that an individual will develop a disease or disorder, eg, a lung disorder. means. The likelihood of developing a disease or disorder is, for example, compared to a population in which individuals with one or more risk factors for the disease or disorder have the same risk factors but have not received the treatment described herein. Thus, statistically speaking, it does not develop a disorder, or decreases when such a disease or disorder is later developed, or at a lower severity. Does not develop symptoms of the disease, or reduces the onset of symptoms (eg, at least 10% on a clinically acceptable scale for the disease or disorder) or delays (eg, days, weeks, months, or years) ) Is considered effective prevention.

  As used herein, the term “inducing differentiation” refers to a more differentiated cell (eg, human lung progenitor cell or airway expression) from a pluripotent or multipotent stem cell (eg, anterior foregut endoderm cells). Means a chemical / biological treatment, body environment, or genetic modification that induces the formation of cells having a type). Differentiation can be assessed by the emergence of distinct cell type specific markers or by the loss of stem cell specific markers, or both.

  As used herein, the terms “comprising” or “comprises” are essential to the present invention, accepting the inclusion of unspecified elements, whether essential or not. Used in connection with certain compositions, methods, and their respective components.

  As used herein, the term “consisting essentially of” means the elements required for a given embodiment. This term permits the presence of additional elements that do not substantially affect the basic and novel or functional features of that aspect of the invention.

  The term “consisting of” means the compositions, methods, and respective components described herein, excluding any element not cited in that description of the embodiment.

  As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” refer to cases where the text clearly indicates otherwise. Excludes plural forms. Thus, for example, reference to “a method” includes one or more methods and / or steps of the type described herein, and others that will be apparent to one of ordinary skill in the art upon reading this disclosure. .

Embryonic stem cells Stem cells are cells that retain the ability to regenerate themselves through mitotic cell division and can differentiate into a diverse range of specialized cell types. Three broad types of mammalian stem cells include embryonic stem (ES) cells found in blastocysts, induced pluripotent stem cells (iPSC) reprogrammed from somatic cells, and adult stem cells found in adult tissues . In developing embryos, stem cells can differentiate into all of the specialized embryonic tissue. In adult organisms, stem and progenitor cells act as a body repair system to recruit specialized cells, but also maintain normal turnover of regenerating organs such as blood, skin, or intestinal tissue . Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.

  Stem cells can, by virtue of their developmental ability, (1) be totipotent, which can give rise to all embryonic and extraembryonic cell types, and (2) give rise to all germ cell types: endoderm, mesoderm, and ectoderm. Can be pluripotent, (3) can be a subset of cell lines, but can be all within a particular tissue, organ, or physiological system (eg, multipotent distal lung progenitors) Cells produce progeny that contain multipotent distal lung progenitor cells (self-renewal) and cell types and elements that are normal components of the airways (eg, basal cells, ciliated cells, Clara cells, and goblet cells) (4) capable of producing a more restricted subset of cell lineages than multipotent stem cells; and (5) can generate one cell lineage (eg, spermatogenic stem cells). Single ability It is classified as.

  Provided herein are methods for producing human lung progenitor cells from both embryonic stem cells and induced pluripotent stem cells. In one embodiment, the methods provided herein relate to the generation of human lung progenitor cells from embryonic stem cells. Alternatively, in some embodiments, the methods provided herein do not include the generation of human lung progenitor cells from embryonic stem cells or any other cell of human embryo origin.

  Embryonic stem cells and methods for their recovery are well known in the art, for example, Trounson AO (Reprod Fertil Dev (2001) 13: 523), Roach ML (Methods Mol Biol (2002) 185: 1), and Smith AG ( Annu Rev Cell Dev Biol (2001) 17: 435). The term “embryonic stem cell” is used to mean a pluripotent stem cell in the inner cell mass of a blastocyst (see, eg, US Pat. Nos. 5,843,780, 6,200,806). Such cells can also be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, eg, US Pat. Nos. 5,955,577, 5,946,619, 6,235,970). The distinguishing characteristics of embryonic stem cells define the embryonic stem cell phenotype. Thus, a cell has an embryonic stem cell phenotype if it has one or more of the unique characteristics of an embryonic stem cell that can distinguish it from other cells. Exemplary embryonic stem cell identification features include, but are not limited to, gene expression profiles, proliferation ability, differentiation ability, karyotype, responsiveness to specific culture conditions, and others.

  Cells derived from embryonic origin can include embryonic stem cells or stem cell lines obtained from stem cell banks or other recognized depositories. Other means of producing stem cell lines include methods involving the use of blastomeres from early embryos (approximately the 8-cell stage) prior to forming blastocysts. Such techniques correspond to preimplantation genetic diagnostic techniques routinely performed at fertility clinics. When one blastomere cell is co-cultured with an established ES cell line and then separated from them, a completely competent ES cell line is formed.

  Embryonic stem cells are considered undifferentiated if they are not tied to a specific differentiation lineage. Such cells exhibit morphological features that distinguish them from differentiated cells of embryonic or adult origin. Undifferentiated embryonic stem (ES) cells are readily recognized by those skilled in the art and typically appear as cell colonies with a high nucleus / cytoplasm ratio and a pronounced nucleolus in a two-dimensional field of microscope. In some embodiments, the human lung progenitor cells described herein are not derived from embryonic stem cells or cells of any other embryonic origin.

  Adult stem cells are stem cells derived from the tissues of an organism after birth or after the neonatal period or from adult organisms, and these are also known in the art. Adult stem cells structurally differ from embryonic stem cells in that there are epigenetic differences, such as differences in DNA methylation patterns, as well as markers that are expressed or not expressed compared to embryonic stem cells.

Induced pluripotent stem cells (iPSC)
In some embodiments, the human lung progenitor cells described herein are derived from isolated pluripotent stem cells. An advantage of using iPSC is that cells can be derived from the same subject to which human lung progenitor cells are administered. That is, somatic cells can be obtained from a subject, reprogrammed into induced pluripotent stem cells, then redifferentiated into human lung progenitor cells (eg, autologous cells) and administered to the subject. Because lung progenitor cells are derived essentially from autologous origin, the risk of engraftment rejection or allergic reaction is reduced compared to using cells from another subject or group of subjects. In some embodiments, the lung progenitor cells are derived from non-autologous origin. In addition, using iPSC eliminates the need to obtain cells from embryonic origin. Thus, in one embodiment, the stem cell used in the disclosed method is not an embryonic stem cell.

  Although differentiation is generally irreversible under physiological conditions, several methods have recently been developed to reprogram somatic cells into induced pluripotent stem cells. Exemplary methods are known in the art and are briefly described herein below.

  As used herein, the term “reprogramming” refers to the process of changing or reversing the differentiation state of a differentiated cell (eg, a somatic cell). In other words, reprogramming refers to the process that drives cell differentiation back to a more undifferentiated or more primitive type of cell. It should be noted that when culturing many primary cells, some of the fully differentiated features can be lost. Thus, simple culture of such cells within the term differentiated cells does not make these cells non-differentiated cells (eg, undifferentiated cells) or pluripotent cells. The transition of differentiated cells to pluripotency requires reprogramming stimuli beyond those that lead to partial loss of differentiation characteristics in culture. Reprogrammed cells are also characterized in that they can be passaged for a long period of time without losing their growth potential, as compared to parent cells of primary cultures that generally have a finite number of divisions in culture.

  The cells to be reprogrammed can be partially differentiated or terminally differentiated prior to reprogramming. In some embodiments, reprogramming includes complete reversal of the differentiated state of a differentiated cell (eg, a somatic cell) to a pluripotent or multipotent state. In some embodiments, reprogramming includes complete or partial reversal of the differentiated state of a differentiated cell (eg, a somatic cell) to an undifferentiated cell (eg, an embryoid-like cell). Reprogramming can cause the expression of certain genes by the cell, and that expression contributes to further reprogramming. In certain embodiments described herein, reprogramming of differentiated cells (eg, somatic cells) can cause differentiated cells to pretend to be undifferentiated (eg, are undifferentiated cells). The obtained cells are called “reprogrammed cells” or “induced pluripotent stem cells (iPSCs or iPS cells)”.

  Reprogramming may involve at least some changes such as reversal, such as genetic patterns of nucleic acid modifications (eg, methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc. that occur during cell differentiation. Reprogramming differs from simply maintaining the existing undifferentiated state of cells that are already pluripotent, or the existing incomplete differentiation state of cells that are already multipotent cells (eg, hematopoietic stem cells). It is also different from maintaining. Although reprogramming is also different from promoting self-renewal or proliferation of cells that are already pluripotent or multipotent, the compositions and methods described herein also include, in some embodiments, their It can also be useful for such purposes.

  The specific approach or method used to generate pluripotent stem cells from somatic cells (generally referred to as “reprogramming”) is not critical to the claimed invention. Thus, any method of reprogramming somatic cells to a pluripotent phenotype would be suitable for use in the methods described herein.

  A reprogramming methodology for generating pluripotent cells using a predetermined combination of transcription factors has been described in induced pluripotent stem cells. Yamanaka and Takahashi transformed mouse somatic cells into ES cell-like cells with increased developmental potential by direct transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006). iPSCs are similar to ES cells because they restore many of the pluripotency-related transcriptional circuits and epigenetic background. In addition, mouse iPSCs are all standard assays for pluripotency, specifically in vitro differentiation into 3 germ layer cell types, teratoma formation, involvement in chimeras, germline transmission (Maherali and Hochedlinger, 2008), and Meet tetraploid blastocyst complementation (Woltjen et al., 2009).

  From subsequent experiments, human iPS cells can be obtained using similar transduction methods (Lowry et al., 2008; Park et al., 2008; Takahashi et al., 2007; Yu et al., 2007b) And the transcription factors trio, OCT4, SOX2, and NANOG have been established as a core set of transcription factors that govern pluripotency (Jaenisch and Young, 2008). Historically, iPS cells can be produced by introducing a nucleic acid sequence encoding a stem cell-related gene into an adult somatic cell using a viral vector.

  iPS cells can be generated or derived from terminally differentiated somatic cells, and can also be generated or derived from adult somatic cells or stem cells. That is, non-pluripotent progenitor cells can be made pluripotent or multipotent by reprogramming. In such instances, it is not necessary to include as many reprogramming factors as are necessary to reprogram the terminally differentiated cells. Furthermore, reprogramming is a messenger that produces a reprogramming factor by introducing a non-viral reprogramming factor, for example by introducing the protein itself, or by introducing a nucleic acid encoding the reprogramming factor, or during translation. It can be induced by introducing RNA (see, for example, Warren et al., Cell Stem Cell, 2010 Nov 5; 7 (5): 618-30). Reprogramming is, for example, Oct-4 (also known as Oct-3 / 4 or Pouf51), Sox1, Sox2, Sox3, Sox15, Sox18, NANOG, Klf1, Klf2, Klf4, Klf5, NR5A2, c-Myc, This can be done by introducing a combination of nucleic acids encoding stem cell associated genes, including l-Myc, n-Myc, Rem2, Tert, and LIN28. In one embodiment, reprogramming using the methods and compositions described herein introduces one or more of Oct-3 / 4, Sox family member, Klf family member, and Myc family member into somatic cells. The method may further include the step of: In one embodiment, the methods and compositions described herein further comprise introducing one or more of each of Oct4, Sox2, Nanog, c-MYC, and Klf4 for reprogramming. . As noted above, the method itself used for reprogramming is not necessarily critical to the methods and compositions described herein. However, when cells differentiated from reprogrammed cells are used, eg, for human therapy, in one embodiment, reprogramming is not performed by methods that alter the genome. Thus, in such embodiments, reprogramming is performed without using, for example, a virus or plasmid vector.

  The efficiency of reprogramming derived from the starting cell population (ie, the number of reprogrammed cells) was calculated using Shi, Y., et al (2008) Cell-Stem Cell 2: 525-528, Huangfu, D., et al (2008) Nature Biotechnology 26 (7): 795-797, and Marson, A., et al (2008) Cell-Stem Cell 3: enhanced by the addition of various small molecules, as shown by 132-135 be able to. Thus, substances or combinations of substances that enhance the efficiency or rate of induced pluripotent stem cell production can be used for the production of patient-specific or disease-specific iPSCs. Some non-limiting examples of substances that enhance reprogramming efficiency include, among others, soluble Wnt, Wnt conditioned media, BIX-01294 (G9a histone methyltransferase), PD0325901 (MEK inhibitor), DNA methyltransferase inhibitor, Histone deacetylase (HDAC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA).

  Other non-limiting examples of reprogramming enhancers include suberoylanilide hydroxamic acid (SAHA (eg, MK0683, vorinostat) and other hydroxamic acids), BML-210, depudecin (eg (-)-depudecine) HC toxin, Nullscript (Nullscript, 4- (1,3-Dioxo-1H, 3H-benzo [de] isoquinolin-2-yl) -N-hydroxybutanamide), phenylbutyrate (eg, sodium phenylbutyrate) And valproic acid ((VPA) and other short chain fatty acids), scriptatide, suramin sodium, trichostatin A (TSA), APHA compound 8, apicidin, sodium butyrate, pivaloyloxymethylbutyrate (Pivanex), AN-9), trapoxin B, clamidosine, depsipeptide (also known as FR901228 or FK228), benzamide (eg CI- 994 (eg, N-acetyldinarine) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA (m-carboxycinnamic acid bishydroxamic acid), JNJ16241199, Tubacin, A-161906, Proxamide Oxamflatin, 3-Cl-UCHA (eg 6- (3-chlorophenylureido) caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50 It is done. Other reprogramming enhancers include, for example, dominant negative forms of HDAC (eg, catalytically inactive forms), siRNA inhibitors of HDAC, and antibodies that specifically bind to HDAC. Such inhibitors are available, for example, from BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Aton Pharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene, and Sigma Aldrich.

  To confirm the induction of pluripotent stem cells for use with the methods described herein, isolated clones can be tested for expression of stem cell markers. If a stem cell marker is expressed in a cell derived from a somatic cell, the cell is identified as an induced pluripotent stem cell. The stem cell marker can be selected from the non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. In one embodiment, cells that express Oct4 or Nanog are identified as pluripotent. Methods for detecting the expression of such markers can include, for example, RT-PCR, and immunological methods that detect the presence of the encoded polypeptide, such as Western blot or flow cytometry analysis. In some embodiments, detection not only involves RT-PCR, but also includes detection of protein markers. Intracellular markers can be best identified by RT-PCR, while cell surface markers are readily identified, for example, by immunocytochemistry.

  The pluripotent stem cell characteristics of the isolated cells can be confirmed by a test that evaluates the differentiation ability of iPSC into each cell of the three germ layers. As an example, teratoma formation in nude mice can be used to evaluate the pluripotency characteristics of isolated clones. Cells are introduced into nude mice and histology and / or immunohistochemistry is performed on tumors arising from the cells. The growth of a tumor that contains cells from all three germ layers further indicates, for example, that the cells are pluripotent stem cells.

  Somatic cells for reprogramming: Somatic cells, as the term is used herein, means any cell that forms the body of an organism, except germline cells. Any cell type in the mammalian body is a differentiated somatic cell, except the sperm and ovum, the cell from which the sperm and ovum are produced (genital cell), and undifferentiated stem cells. For example, internal organs, skin, bone, blood, and connective tissue are all composed of differentiated somatic cells.

  Additional somatic cell types used with the compositions and methods described herein are fibroblasts (eg, primary cultured fibroblasts), muscle cells (eg, myocytes), hill cells, neurons, mammary cells. , Including hepatocytes and islet cells. In some embodiments, the somatic cell is a primary cell line or a progeny of a primary or secondary cell line. In some embodiments, somatic cells are obtained from a human sample, eg, from a hair follicle, blood sample, biopsy (eg, skin biopsy, or adipose tissue biopsy), swab sample (eg, oral swab sample). Because of this, it is a human somatic cell.

  Some non-limiting examples of differentiated somatic cells include epithelial cells, endothelial cells, neurons, fat cells, heart cells, skeletal muscle cells, immune cells, hepatocytes, spleen cells, lung cells, circulating blood cells , Gastrointestinal cells, kidney cells, bone marrow cells, and pancreatic cells. In some embodiments, somatic cells are from any body tissue, including but not limited to brain, liver, lung, gastrointestinal tract, stomach, intestinal tract, fat, muscle, uterus, skin, spleen, endocrine organ, bone, and the like. It can be an isolated primary culture cell. Furthermore, somatic cells can be obtained from any mammalian species, non-limiting examples of which include mouse, cow, monkey, pig, horse, sheep, or human cells. In some embodiments, the somatic cell is a human somatic cell.

  When using reprogrammed cells to make human lung progenitor cells used for therapeutic treatment of disease, it is desirable, but not necessarily, to use somatic cells isolated from the patient being treated. For example, somatic cells associated with the disease, somatic cells involved in the therapeutic treatment of the disease, and others can be used. In some embodiments, the method of selecting reprogrammed cells from a heterogeneous population comprising reprogrammed cells and the somatic cells from which they are derived or generated can be performed by any known means. . For example, reprogrammed cells can be isolated using a drug resistance gene such as a selectable marker gene or others and using the selectable marker as an indicator.

  The reprogrammed somatic cells disclosed herein include alkaline phosphatase (AP); ABCG2; stage-specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; 1-81; Tra-2-49 / 6E; ERas / ECAT5, E-cadherin; β-III-tubulin; α-smooth muscle actin (α-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); ES cell-related transcript 1 (ECAT1); ESG1 / DPPA5 / ECAT2; ECAT3; ECAT6; ECAT7; ECAT8; ECAT9; ECAT10; ECAT15 -1; ECAT15-2; Fthl17; Sal14; Undifferentiated germ cell transcription factor (Utf1); Rex1; p53; G3PDH; Telomerase including TERT; Silent X chromosome gene; Dnmt3a; Dnmt3b; TRIM28; Fbx15); Nanog / ECAT4; Oct3 / 4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3; CYP25A1; developmental pluripotency-associated 2 (D Any number of pluripotent cell markers can be expressed, including PPA2); T-cell lymphoma break point 1 (Tcl1); DPPA3 / Stella; DPPA4; other general markers for pluripotency. Other markers may include Dnmt3L; Sox15; Stat3; Grb2; β-catenin, and Bmi1. Such cells can also be characterized by marker downregulation that is characteristic of somatic cells from which induced pluripotent stem cells are derived.

Production of definitive endoderm and anterior foregut endoderm The method of producing human lung progenitor cells described herein begins with the production of definitive endoderm from embryonic stem cells or induced pluripotent stem cells. “Definitive endoderm” includes multipotent cells that are restricted to the endoderm lineage and can give rise to intestinal or organs derived from the intestinal tract. Definitive endoderm is the digestive organs (eg, esophagus, stomach, liver, gallbladder, small intestine, pancreas, colon, etc.), respiratory organs (eg, alveoli, trachea, bronchi), endocrine glands and organs (eg, parathyroid, thyroid) , Thymus), auditory system (eg, canal and tympanic) and urinary system (eg, part of the bladder and urethra). See, for example, Grapin-Botton and Melton, 2000; Kimelman and Griffin, 2000; Tremblay et al., 2000; Wells and Melton, 1999; Wells and Melton, 2000. The term “definitive endoderm” does not encompass a different lineage of cells called primordial endoderm that is responsible for the formation of extraembryonic tissue.

  Formation of definitive endoderm and endoderm cells derived therefrom induces cells that make up terminally differentiated tissues and / or organs derived from the definitive endoderm lineage, such as the human lung progenitor cells described herein. Is an important stage for.

  Methods for inducing definitive endoderm from embryonic stem cells or induced pluripotent stem cells are known in the art (eg, US Pat. Nos. 7,993,916; 7,695,963; 7,541,185; US2009 / 0298178; US2010 / 0272695; Sherwood et al., Mechanisms of Development (2011) 128: 387-400; D'Amour K. et al., Nature Biotechnology (2005) 23: 1534-1541; Turovets, N. et al., Differentiation (2011) 81 (5): 292-298; see Kim, PT. Et al., PLoS One (2010) 5 (11): e14146). In one embodiment, definitive endoderm is produced by contacting IPSC or ESC with definitive endoderm medium comprising, for example, B27, activin A and ZSTK474. In one embodiment, the definitive endoderm medium comprises 1-5% B27 (eg, 2%), 10-40 ng / mL activin A (eg, 20 ng / mL), and 0.2-0.5 μM ZSTK474 and tested Thus, it is useful for producing definitive endoderm in a cell line suspected of having low definitive endoderm production efficiency or low definitive endoderm production efficiency.

  Differentiation of embryonic stem cells or induced pluripotent stem cells into definitive endoderm can be monitored by determining the expression of cell surface markers that are characteristic of definitive endoderm. In some embodiments, the expression of definitive endoderm markers is determined by detecting the presence or absence of the markers. Alternatively, the expression of a marker can be determined by measuring the level at which the marker is present in cells in a cell culture or cell population. Such measurement of marker expression can be either qualitative or quantitative.

  In one embodiment, quantitative PCR (Q-PCR) is used to quantify the expression of the marker in the definitive endoderm. Methods for performing Q-PCR are well known in the art. In an alternative embodiment, an antibody specific for a cellular marker is used to detect the expression of the marker gene product. In certain embodiments, the expression of marker genes that are characteristic of definitive endoderm and the lack of significant expression of marker genes that are characteristic of the cells from which they are derived (eg, ES cells or iPSCs) and other cell types. To decide.

  In one embodiment, the definitive endoderm marker is the SOX17 gene. Other markers of definitive endoderm include, but are not limited to, MIXL1, GATA4, HNF3b, GSC, FGF17, VWF, CALCR, FOXQ1, CMKOR1, and CRIP1. In some embodiments, both SOX17 and SOX7 expression is monitored. In other embodiments, the expression of the SOX17 marker gene and the OCT4 marker gene characteristic of ES cells is monitored. Furthermore, since definitive endoderm cells express SOX17 marker genes at higher levels than AFP, SPARC, or thrombomodulin marker genes, the expression of these genes can be monitored as well. Another marker of definitive endoderm is the CXCR4 gene, which encodes a cell surface chemokine receptor whose ligand is chemotactic SDF-1. In one embodiment, definitive endoderm production efficiency can be determined by FACS analysis with FOXA2 / SOX17 co-staining or a combination of cKit / CXCR4 or cKit / EpCAM.

  After the definitive endoderm has been created, the next step is to differentiate the definitive endoderm cells into anterior foregut endoderm cells, a region containing cells destined to become lung and thyroid cells. . This process is also referred to herein as “forward movement” of the definitive endoderm. “Foregut endoderm” as used herein refers to cells in the anterior part of the intestine and includes cells in the foregut / midgut junction. It will be appreciated by those skilled in the art that ESCs or iPSCs can also be differentiated directly into anterior foregut endoderm cells without the need for an intermediate step to produce definitive endoderm. The differentiation methods described herein for producing lung progenitor cells begin with anterior foregut endoderm cells, and thus the method for producing such anterior foregut endoderm cells is not important and this Not limited to the methods of making anterior foregut endoderm described in the specification; any method that provides for anterior foregut endoderm is used to initiate the preparation of lung progenitor cells disclosed herein Material can be provided.

  Methods for producing anterior foregut endoderm from definitive endoderm are known in the art (eg, WO2010 / 136583, WO2011 / 139628; Green, MD et al., Nature Biotechnology (2011) 29: 267-27 Morrison et al, (2008), Cell Stem Cell, 3: 355-356; Goss AM et al., Developmental Cell (2009) 17 (2): 290-298; Livigni A et al., Current Protocols in Stem Cell Biology (2009) 10: See 1G.3.1-1G.3.10).

  In one embodiment, production of anterior foregut endoderm is confirmed by activation of an anterior foregut endoderm specific marker, such as marker Hex. Hex is one of the earliest markers of the anterior foregut endoderm and is a homeobox-containing transcriptional repressor that has been shown to suppress posterior features (eg, Brickman JM et al., Development (2000 127: 2303-2315; Thomas PQ et al., Development (1998) 125: 85-94; see Zamparini AL et al., Development (2006) 133: 3709-3722). Hex detection can be used in combination with other anterior foregut endoderm markers such as Cxcr4 (Morrison, GM et al., Cell Stem Cell (2008) 3: 402-412). Other exemplary markers include, but are not limited to, FoxA2 and Sox2. In one embodiment, the definitive endoderm undergoes a forward migration phase that includes treatment with a TGFβ agonist (eg, activin).

Signaling pathway for differentiation
Modulation of the TGF-β signaling pathway: In some embodiments, one or more TGF- is selected to promote specific differentiation stages of pluripotent cells (eg, during the production of anterior foregut endoderm). Beta agonists are used. In such embodiments, the activator specific for TGF-β signaling is TGF-β polypeptide or an active fragment thereof, a fusion protein comprising a TGF-β polypeptide or an active fragment thereof, TGF-β receptor It can be an agonist antibody to the body, or a small molecule agonist of the TGF-β receptor.

  In other embodiments, one or more TGF-β antagonists can be used to differentiate pluripotent cells (eg, a first stage that is restricted to the lung lineage to induce Nkx2.1 expression). . In such embodiments, the antagonist of TGF-β signaling can be a polypeptide inhibitor or fragment thereof, a dominant negative fusion protein, an antagonist antibody to the TGF-β receptor, or a small molecule antagonist of the TGF-β receptor. .

  The transforming growth factor beta (TGF-beta) signaling pathway is a number of cellular processes in both adult organisms and developing embryos, including cell growth, cell differentiation, apoptosis, cell homeostasis, and other cellular functions Is related to. TGF-β superfamily ligands bind to type II receptors, which recruit and phosphorylate type I receptors. The type I receptor then phosphorylates receptor-modulated SMAD (R-SMAD), which in turn binds to coSMAD SMAD4. R-SMAD / coSMAD complexes accumulate in the nucleus where they act as transcription factors and are involved in the regulation of target gene expression.

  TGF-β1 is a prototypical member of the cytokine family that includes TGF-β, activin, inhibin, bone morphogenetic protein, and Müller tube inhibitor. The Smad protein is an exemplary downstream signaling factor in the TGF-β pathway, and thus in some embodiments can be directly activated to undergo differentiation into the human lung cell precursor phenotype ( (For example, by treating cells with an activator of the Smad protein). Exemplary Smad activators include, but are not limited to, Smad protein or functional peptides or fragments thereof (eg, Smad1, Smad5, Smad8), BMP2, BMP4, and Muller tube inhibitor (MIS) Do not mean. Activin ligands transmit signals in a manner similar to TGFβ ligands. Activin binds to and activates the ALK receptor and then phosphorylates Smad proteins such as Smad2 and Smad3. Subsequent formation of a hetero-Smad complex with Smad4 results in activin regulation of gene transcription.

  Some non-limiting examples of small molecule inhibitors of the TGF-β receptor include 2- (3- (6-methylpyridin-2-yl) -1H-pyrazol-4-yl) -1,5 Naphthyridine, [3- (pyridin-2-yl) -4- (4-quinoyl)]-1H-pyrazole, and 3- (6-methylpyridin-2-yl) -4- (4-quinolyl) -1- And phenylthiocarbamoyl-1H-pyrazole, which can be purchased from Calbiochem (San Diego, Calif.). Other small molecule inhibitors include SB-431542 (see, eg, Haider et al., 2005; Neoplasia 7 (5): 509-521), SM16 (eg, Fu, K et al., 2008). Arteriosclerosis, Thrombosis and Vascular Biology 28 (4): 665); and SB-505124 (see, eg, Dacosta Byfield, S., et al., 2004; Molecular Pharmacology 65: 744-52) However, it is not limited to these. Additional TGF-β receptor antagonists are known in the art.

  In some embodiments, useful dosage ranges for TGF-β antagonists (eg, A8301) are 0.1 to 10 μM, such as 0.1 to 1 μM, 0.1 to 0.5 μM, 0.1 to 2 μM, 0.1 to 3 μM, 0.1 to 4 μM, 0.1 to 5 μM, 0.1 to 6 μM, 0.1 to 7 μM, 0.1 to 8 μM, 0.1 to 9 μM, 0.5 to 2 μM, 0.5 to 5 μM, 1 to 3 μM, 2 to 4 μM, 2 to 6 μM, 2 to 7 μM, 5 to 10 μM, 6 to 10 μM, 7 to 10 μM, 8 to 10 μM, 9 to 10 μM. In some embodiments, the TGF-β antagonist is, for example, at least 0.1 μM, at least 0.2 μM, at least 0.3 μM, at least 0.4 μM, at least 0.5 μM, at least 0.6 μM, at least 0.7 μM, at least 0.8 μM, at least 0.9 μM, at least 1 μM, at least 1.2 μM, at least 1.3 μM, at least 1.4 μM, at least 1.5 μM, at least 1.6 μM, at least 1.7 μM, at least 1.8 μM, at least 1.9 μM, at least 2 μM, at least 2.5 μM, at least 3 μM, at least 3.5 μM, at least 4 μM , At least 4.5 μM, at least 5 μM, at least 5.5 μM, at least 6 μM, at least 6.5 μM, at least 7 μM, at least 7.5 μM, at least 8 μM, at least 8.5 μM, at least 9 μM, at least 9.5 μM, at least 10 μM or more .

Regulation of BMP receptor signaling pathway
Both BMP2 and BMP4 transduce signals through the type I receptor (ALK3), while BMP7 binds to a different type I receptor (ALK2). For example, von Bubnoff A et al., Developmental Biology (2001) 239: 1-14; Chen D. et al., Growth Factors (2004) 22 (4): 233-241; Sieber C. et al., Cytokine and See Growth Factor Rev. (2009) 20: 343-355; and Miyazono K et al., Journal of Biochemistry (2010) 147 (1): 35-51.

  Typically, BMP2 and BMP4 bind to the BMP receptor I / II complex, which results in phosphorylation of Smads 1/5/8, followed by the formation of a heterotrimeric complex with Smad4 . These complexes migrate to the nucleus and activate target gene expression (von Bubnoff A et al., Developmental Biology (2001) 239: 1-14; Chen D. et al., Growth Factors (2004) 22 (4): 233-241; Sieber C. et al., Cytokine and Growth Factor Rev. (2009) 20: 343-355; and Miyazono K et al., Journal of Biochemistry (2010) 147 (1): 35 -51). In addition to Smad1 / 5 / 8-mediated transcription, BMP-induced receptor complexes can activate the mitogen-activated protein kinase (MAPK) pathway via ERK, JNK, or p38 (Kozawa O et al. , Journal of Cellular Biochemistry 84: 583-589).

  BMP receptor pathway activation: In some embodiments, BMP agonists are used with the methods described herein to differentiate human lung progenitor cells. In one embodiment, the BMP receptor is a receptor that transmits signals through the SMAD pathway (eg, ALK3). In other embodiments, the BMP used with the methods described herein is BMP2 and / or BMP4.

  In one embodiment, one or more BMP agonists are used to promote specific differentiation stages of pluripotent cells. In such embodiments, the activator specific for BMP signaling is a BMP polypeptide or active fragment thereof, a fusion protein comprising a BMP polypeptide or active fragment thereof, an agonist antibody to a BMP receptor, or BMP receptor. It can be a small molecule agonist of the body.

  In some embodiments, useful dose ranges for BMP4 are 1 to 500 nM, such as 1 to 400 nM, 1 to 300 nM, 1 to 200 nM, 1 to 100 nM, 1 to 50 nM, 1 to 25 nM, 1 To 10 nM, 1 to 5 nM, 1 to 2 nM, 10 to 300 nM, 15 to 250 nM, 20 to 250 nM, 20 to 200 nM, 30 to 200 nM, 40 to 200 nM, 50 to 200 nM, 60 To 200 nM, 70 to 200 nM, 80 to 200 nM, 90 to 200 nM, 100 to 200 nM, 150 to 200 nM, 150 nM to 300 nM, 175 to 300 nM, 200 nM to 300 nM, 200 nM to 400 nM, 200 nM to 500 nM.

  In some embodiments, the dose of BMP4 is, for example, at least 1 nM, at least 2 nM, at least 5 nM, at least 10 μM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 60 nM, at least 70 nM. At least 80 nM, at least 90 nM, at least 100 nM, at least 110 nM, at least 120 nM, at least 130 nM, at least 140 nM, at least 150 nM, at least 160 nM, at least 170 nM, at least 180 nM, at least 190 nM, at least 200 nM, at least 225 nM, at least 250 nM, at least 275 nM, at least 300 nM, at least 400 nM, at least 500 nM or more.

  In some embodiments, useful dosage ranges for BMP7 are 1 to 200 ng / mL, such as 1 to 100 ng / mL, 1 to 50 ng / mL, 1 to 25 ng / mL, 1 to 10 ng / mL, 1 to 5 ng / mL, 1 to 2 ng / mL, 10 to 200 ng / mL, 15 to 200 ng / mL, 20 to 200 ng / mL, 30 to 200 ng / mL, 40 to 200 ng / mL, 50 To 200 ng / mL, 60 to 200 ng / mL, 70 to 200 ng / mL, 80 to 200 ng / mL, 90 to 200 ng / mL, 100 to 200 ng / mL, or 150 to 200 ng / mL .

  In some embodiments, the dose of BMP7 is, for example, at least 1 ng / mL, at least 2 ng / mL, at least 5 ng / mL, at least 10 ng / mL, at least 20 ng / mL, at least 30 ng / mL, at least 40 ng / mL, at least 50 ng / mL, at least 60 ng / mL, at least 70 ng / mL, at least 80 ng / mL, at least 90 ng / mL, at least 100 ng / mL, at least 110 ng / mL, at least 120 ng / mL, at least 130 ng / mL, at least 140 ng / mL, at least 150 ng / mL, at least 160 ng / mL, at least 170 ng / mL, at least 180 ng / mL, at least 190 ng / mL, at least 200 ng / mL, Or more.

  Inhibition of the BMP receptor pathway: In some embodiments, BMP antagonists are used with the methods described herein to differentiate human foregut endoderm cells into lung progenitor cells. In one embodiment, the BMP antagonist is dorsomorphin.

  In one embodiment, one or more BMP receptor pathway antagonists are used to promote specific differentiation stages of pluripotent cells. In such embodiments, the inhibitor specific for BMP signaling is a polypeptide or fragment thereof, an shRNA or siRNA against the BMP receptor, an antagonist antibody against the BMP receptor, or a small molecule antagonist of the BMP receptor. sell.

  In some embodiments, useful dosage ranges for BMP pathway inhibitors are 1 to 500 nM, such as 1 to 400 nM, 1 to 300 nM, 1 to 200 nM, 1 to 100 nM, 1 to 50 nM, 1 to 25 nM, 1 to 10 nM, 1 to 5 nM, 1 to 2 nM, 10 to 300 nM, 15 to 250 nM, 20 to 250 nM, 20 to 200 nM, 30 to 200 nM, 40 to 200 nM, 50 to 200 nM, 60 to 200 nM, 70 to 200 nM, 80 to 200 nM, 90 to 200 nM, 100 to 200 nM, 150 to 200 nM, 150 nM to 300 nM, 175 to 300 nM, 200 nM to 300 nM, 200 nM to 400 nM, 200 nM to 500 nM.

  In some embodiments, the dose of the BMP pathway antagonist is, for example, at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 60 nM, At least 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, at least 110 nM, at least 120 nM, at least 130 nM, at least 140 nM, at least 150 nM, at least 160 nM, at least 170 nM, at least 180 nM, at least 190 nM, at least 200 nM, at least 225 nM, at least 250 nM, at least 275 nM, at least 300 nM, at least 400 nM, at least 500 nM or more.

  MAPKK ERK inhibitor: As used herein, human lung progenitor cells, including treatment with MAPKK / ERK inhibitors, are treated as Nkx2.1 + Sox2 + proximal multipotent airway progenitor cells or Nkx2.1 + Sox9 + distal multipotent A method for differentiating into progenitor cells is provided.

Mitogen-activated protein kinase (MAPK) signaling pathways have been implicated in cellular events such as growth, differentiation, and stress response (J. Biol. Chem. (1993) 268, 14553-14556). To date, four similar MAPK pathways have been identified: ERK1 / ERK2, JNK, p38, and ERK5. These pathways are linear kinase cascades in that MAPKKK phosphorylates and activates MAPKK, and MAPKK phosphorylates and activates MAPK. To date, seven MAPKK homologs (MEK1, MEK2, MKK3, MKK4 / SEK, MEK5, MKK6, and MKK7) and four MAPK families (ERKl / 2, JNK, p38, and ERK5) have been identified . Activation of these pathways regulates the activity of numerous substrates through phosphorylation. These substrates include TCF, c-myc, ATF2, and AP-1 components, transcription factors such as fos and Jun; cell surface components EGF-R; PHAS-T, p90 ^rsk , ^cPLA ₂ and c-Raf-1. Including cytoplasmic components; and cytoskeletal components such as tau and MAP2. The MAPK signaling cascade is involved in the regulation of cellular processes including proliferation, differentiation, apoptosis, and stress response.

  MEK occupies a strategic downstream position in the Mek / Erk pathway that catalyzes phosphorylation of its MAPK substrates ERK1 and ERK2. Anderson et al. Nature 1990, v.343, pp. 651-653. In the ERK pathway, MAPKK corresponds to MEK (MAP kinase ERK kinase), and MAPK corresponds to ERK (extracellular regulatory kinase).

  Some non-limiting examples of MAPK and / or ERK pathway inhibitors include SL327, U0126, SP600125, PD98059, SB203580, and CAY10561. Additional MAPK and / or ERK pathway inhibitors that can be used with the methods described herein are known to those of skill in the art.

  In some embodiments, useful dose ranges for MAPKK / ERK antagonists (eg, PD98059) are 0.1 to 5 μM, such as 0.1 to 4 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1 μM, 0.1 to 0.5 μM, 0.5 To 3 μM, 0.5 to 2 μM, 0.5 to 1 μM, 1 to 2 μM, 1.5 to 2 μM, 1 to 1.5 μM, 2 to 5 μM, 3 to 5 μM, 4 to 5 μM.

  In some embodiments, the dose of the MAPKK / ERK antagonist is, for example, at least 0.1 μM, at least 0.5 μM, at least 1 μM, at least 1.1 μM, at least 1.2 μM, at least 1.3 μM, at least 1.4 μM, at least 1.5 μM, at least 1.6 μM, At least 1.7 μM, at least 1.8 μM, at least 1.9 μM, at least 2 μM, at least 2.5 μM, at least 3 μM, at least 4 μM, at least 5 μM or more.

  FGF activation: Fibroblast growth factor, or FGF, is a family of growth factors that play a role in angiogenesis, wound healing, and embryonic development. FGF and functional fragments or analogs thereof, as described herein, include human lung progenitor cells such as proximal multipotent airway progenitor cells, distal multipotent lung progenitor cells, and multipotent airway bases Useful for differentiating into stem cells.

  FGF is a heparin-binding protein that interacts with cell surface-associated heparan sulfate proteoglycans to perform FGF signaling. At least 22 different members of the FGF family have been identified. FGFl, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, and FGF10 bind through the fibroblast growth factor receptor (FGFR) to perform signal transmission.

  FGF induces mitosis in a variety of cell types and has regulatory, morphological, and endocrine effects as well. FGFs function throughout embryonic development to help mesoderm induction, anterior-posterior patterning, leg development, nerve induction, and nerve development. In one embodiment, a preferred FGF for use with the methods described herein is FGF7, which is also known as keratinocyte growth factor (KGF).

  In some embodiments, useful dose ranges for FGF7 or FGF2 are 10 to 200 ng / mL, such as 10 to 100 ng / mL, 10 to 50 ng / mL, 15 to 200 ng / mL, 20 to 200 ng / mL. mL, 30 to 200 ng / mL, 40 to 200 ng / mL, 50 to 200 ng / mL, 60 to 200 ng / mL, 70 to 200 ng / mL, 80 to 200 ng / mL, 90 to 200 ng / mL 100 to 200 ng / mL, or 150 to 200 ng / mL.

  In some embodiments, the dose of FGF7 or FGF2 is, for example, at least 10 ng / mL, at least 20 ng / mL, at least 30 ng / mL, at least 40 ng / mL, at least 50 ng / mL, at least 60 ng / mL, At least 70 ng / mL, at least 80 ng / mL, at least 90 ng / mL, at least 100 ng / mL, at least 110 ng / mL, at least 120 ng / mL, at least 130 ng / mL, at least 140 ng / mL, at least 150 ng / mL, at least 160 ng / mL, at least 170 ng / mL, at least 180 ng / mL, at least 190 ng / mL, at least 200 ng / mL, at least 225 ng / mL, at least 250 ng / mL, or more .

  Modulation of the Wnt pathway: Without wishing to be bound by theory, the Wnt protein and its cognate receptor transmit signals through at least two distinct intracellular pathways. The “canonical” Wnt signaling pathway (referred to herein as the Wnt / β-catenin pathway) involves Wnt signaling via β-catenin to activate transcription through TCF-related proteins (van de Wetering et al. (2002) Cell 109 Suppl: S13-9; Moon et al. (2002) Science 296 (5573): 1644-6). There are non-standard alternative pathways by which Wnt activates protein kinase C (PKC), calcium / calmodulin-dependent kinase II (CaMKII), JNK, and Rho-GTPase (Veeman et al. (2003) Dev Cell 5 (3): 367-77), often associated with cell polarity control.

  Wnt antagonist: As used herein, by contacting a cell with a Wnt antagonist, a human lung progenitor cell is transformed into a more differentiated stem cell phenotype, eg, Nkx2.1 + Sox2 + proximal multipotent airway progenitor cell or Nkx2.1 + Methods are provided for differentiation into Sox9 + distal multipotent lung progenitor cells, or Nkx2.1 + p63 + multipotent airway basal stem cells.

  As used herein, the term “Wnt antagonist” or “Wnt inhibitor” is any that inhibits the Wnt / β-catenin pathway or enhances the activity and / or expression of an inhibitor of Wnt / β-signaling. Or an activator or enhancer of GSK-3β activity. As used herein, Wnt inhibitors include, for example, Wnt or β-catenin, or decreased Wnt-dependent gene and / or protein expression and / or activity, and endogenous inhibitors of Wnt and / or β-catenin. Pathways due to, but not limited to, increased expression and / or activity, or increased expression and / or activity of endogenous inhibitors of components of the Wnt / β-catenin pathway, such as but not limited to increased expression of GSK-3β Can inhibit the Wnt / β-catenin pathway at any point.

  Some non-limiting examples of Wnt antagonists include the Wnt pathway inhibitor V (also known as (E) -4- (2,6-difluorostyryl) -N, N-dimethylaniline), IWR-1 and peptides containing endo, IWP-2, CCT036477, and the sequence t-Boc-NH-Met-Asp-Gly-Cys-Glu-Leu-C02H (SEQ ID NO: 1).

  In some embodiments, useful dosage ranges for Wnt antagonists (eg, IWR-1) are 20 to 200 ng / mL, 30 to 200 ng / mL, 40 to 200 ng / mL, 50 to 200 ng / mL, 60 to 200 ng / mL, 70 to 200 ng / mL, 80 to 200 ng / mL, 90 to 200 ng / mL, 100 to 200 ng / mL, or 150 to 200 ng / mL.

  In some embodiments, the dose of the Wnt antagonist is, for example, at least 20 ng / mL, at least 30 ng / mL, at least 40 ng / mL, at least 50 ng / mL, at least 60 ng / mL, at least 70 ng / mL, at least 80 ng / mL, at least 90 ng / mL, at least 100 ng / mL, at least 110 ng / mL, at least 120 ng / mL, at least 130 ng / mL, at least 140 ng / mL, at least 150 ng / mL, at least 160 ng / mL, at least 170 ng / mL, at least 180 ng / mL, at least 190 ng / mL, at least 200 ng / mL, or more.

  Wnt agonist: As used herein, a method for differentiating human foregut endoderm cells into more differentiated cell types, eg, Nkx2.1 + Tuj negative Pax8 negative lung progenitor cells, by contacting the cell with a Wnt agonist. Provided.

  As used herein, the term “Wnt agonist” refers to any substance that activates the Wnt / β-catenin pathway or inhibits the activity and / or expression of an inhibitor of Wnt / β-catenin signaling, such as Means an antagonist or inhibitor of GSK-3β activity. As used herein, Wnt activators are, for example, Wnt or β-catenin, or increased expression and / or activity of Wnt-dependent genes and / or proteins, and endogenous inhibitors of Wnt and / or β-catenin Decrease in the expression and / or activity of, or decrease in expression and / or activity of endogenous inhibitors of components of the Wnt / β-catenin pathway, such as but not limited to: Signal transduction can be enhanced through the Wnt / β-catenin pathway at any point along the pathway.

  Some non-limiting examples of Wnt pathway agonists include CHIR9902, 2-amino-4- [3,4- (methylenedioxy) benzyl-amino] -6- (3-methoxyphenyl) pyrimidine, BIO, (2'Z, 3'E) -6-bromoindirubin-3'-oxime, 5- (furan-2-yl) -N- (3- (1H-imidazol-1-yl) propyl) -l, 2-oxazole-3-carboxamide, and SKL2001.

  In some embodiments, useful dosage ranges for Wnt agonists (eg, CHIR9902) are 20 to 200 ng / mL, 30 to 200 ng / mL, 40 to 200 ng / mL, 50 to 200 ng / mL, 60 to 200 ng / mL, 70 to 200 ng / mL, 80 to 200 ng / mL, 90 to 200 ng / mL, 100 to 200 ng / mL, or 150 to 200 ng / mL.

  In some embodiments, the dose of the Wnt agonist is, for example, at least 20 ng / mL, at least 30 ng / mL, at least 40 ng / mL, at least 50 ng / mL, at least 60 ng / mL, at least 70 ng / mL, at least 80 ng / mL, at least 90 ng / mL, at least 100 ng / mL, at least 110 ng / mL, at least 120 ng / mL, at least 130 ng / mL, at least 140 ng / mL, at least 150 ng / mL, at least 160 ng / mL, at least 170 ng / mL, at least 180 ng / mL, at least 190 ng / mL, at least 200 ng / mL, or more.

  In some embodiments, useful dosage ranges for Wnt agonists (eg, CHIR9902) are 0.1 to 5 μM, such as 0.1 to 4 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1 μM, 0.1 to 0.5 μM, 0.5 to 3 μM, 0.5 to 2 μM, 0.5 to 1 μM, 1 to 2 μM, 1.5 to 2 μM, 1 to 1.5 μM, 2 to 5 μM, 3 to 5 μM, 4 to 5 μM.

  In some embodiments, the dose of the Wnt agonist (eg, CHIR9902) is, for example, at least 0.1 μM, at least 0.5 μM, at least 1 μM, at least 1.1 μM, at least 1.2 μM, at least 1.3 μM, at least 1.4 μM, at least 1.5 μM, at least 1.6 μM, at least 1.7 μM, at least 1.8 μM, at least 1.9 μM, at least 2 μM, at least 2.5 μM, at least 3 μM, at least 4 μM, at least 5 μM, or more.

  PI3 kinase inhibitor: Phosphoinositide 3-kinase (PI3K) is a lipid kinase that phosphorylates lipids at the 3-hydroxyl residue of the inositol ring (Whitman et al (1988) Nature, 332: 664). 3-phosphorylated phospholipids (PIP3) produced by PI3 kinases recruit two kinases with lipid binding domains (including the plekstrin homology (PH) region), such as Akt and phosphoinositide-dependent kinase 1 (PDK1). Acts as a secondary transmitter. When Akt binds to membrane PIP3, Akt moves to the cytoplasmic membrane, and Akt contacts PDK1, which causes Akt activation. PTEN, a tumor suppressor phosphatase, dephosphorylates PIP3 and therefore acts as a negative regulator of Akt activation. PI3 kinases Akt and PDK1 are important in the regulation of many cellular processes, including cell cycle regulation, proliferation, survival, apoptosis, and mobility, and are responsible for the molecular mechanisms of diseases such as cancer, diabetes, and immune inflammation It is a significant component (Vivanco et al (2002) Nature Rev. Cancer 2: 489; Phillips et al (1998) Cancer 83:41).

  As used herein, the term “PI3 kinase inhibitor” or “PI3 kinase antagonist” means any substance that inhibits the activity of PI3 kinase. Some non-limiting examples of PI3 kinase inhibitors useful by the methods described herein include LY294002, wortmannin, PIK-75, ZSTK474, and Pp242.

  In some embodiments, useful dosage ranges for PI3 kinase inhibitors (eg, ZSTK474, or PIK-75) are 0.1 to 5 μM, such as 0.1 to 4 μM, 0.1 to 3 μM, 0.1 to 2 μM, 0.1 to 1 μM, 0.1 to 0.5 μM, 0.5 to 3 μM, 0.5 to 2 μM, 0.5 to 1 μM, 1 to 2 μM, 1.5 to 2 μM, 1 to 1.5 μM, 2 to 5 μM, 3 to 5 μM, 4 to 5 μM.

  In some embodiments, the dose of a PI3 kinase inhibitor (eg, ZSTK474, or PIK-75) is, for example, at least 0.1 μM, at least 0.5 μM, at least 1 μM, at least 1.1 μM, at least 1.2 μM, at least 1.3 μM, at least 1.4. μM, at least 1.5 μM, at least 1.6 μM, at least 1.7 μM, at least 1.8 μM, at least 1.9 μM, at least 2 μM, at least 2.5 μM, at least 3 μM, at least 4 μM, at least 5 μM, or more.

Monitoring differentiation of human lung progenitor cells As used herein, pluripotent stem cells (eg, anterior foregut endoderm cells, definitive endoderm cells, ES cells, or iPSCs) are differentiated or redifferentiated into human lung progenitor cells. Optionally providing further differentiation of such human lung progenitor cells into lung airway cells such as basal cells, Clara cells, cilia cells, and / or goblet cells. These aspects are based on the newly discovered method of differentiating pluripotent stem cells into stem or progenitor cells that are restricted to the lung and / or airway lineage. Such a method is illustrated in the Examples section herein. Similarly, provided herein are compositions of human lung progenitor cells having certain characteristics, such as the presence of one or more cell surface markers or other markers that are lung cell specific. Alternatively or additionally, the human lung progenitor cell compositions described herein lack a marker for embryonic stem cells or induced pluripotent stem cells. In one embodiment of the methods described herein, one or more cell surface markers are used to determine the degree of differentiation along the spectrum from embryonic stem cells or iPSCs to fully differentiated lung cells.

  Cell surface markers, particularly stem cell surface markers, are useful by the methods and compositions described herein to identify the differentiation or dedifferentiation status of cells. For example, upon reprogramming somatic cells to induced pluripotent stem cells, activation of stem cell markers can be used to confirm that the somatic cells are (partially or completely) dedifferentiated. Alternatively, when ES cells or iPSCs are differentiated into human lung progenitor cells, activation of lung-specific markers can be used to confirm the degree of differentiation that stem cells are undergoing. In addition, activation or deactivation of certain lung specific markers can be used to determine the degree of multipotency of human lung progenitor cells. This compares lung-specific markers present on or expressed by cells with the marker profile of lung cells during development, to a known degree of multipotency of the corresponding lung cells during embryo development Based on gender, this can be done by estimating the degree of multipotency of differentiated cells.

  A stem cell marker can be recognized using a marker-specific substance, such as a labeled antibody that recognizes and binds to a cell surface marker or antigen on a desired stem cell. Using antibodies or similar substances specific for a given marker or set of markers, fluorescence activated cell sorting (FACS), panning method, magnetic particle selection, particle sorter selection, and density separation (Xu et al. (2002) Circ. Res. 91: 501; U.S. Patent Application No. 20030022367) and other physical properties based separation (Doevendans et al. (2000) J. Mol. Cell. Cardiol. 32: 839-851) Other methods known to those skilled in the art can be used to isolate and isolate the desired stem cells.

  Alternatively, gene selection methods can be used when progenitor cells or stem cells can be genetically engineered to express a reporter protein operably linked to a tissue-specific promoter and / or a specific gene promoter. Therefore, reporter expression can be used in the case of a positive selection method to isolate and enrich for the desired stem cells. For example, a fluorescent reporter protein can be expressed in a desired stem cell by genetic engineering to functionally link the marker protein to a promoter that is active in the desired stem cell (Klug et al. (1996) J. Clin Invest. 98: 216-224; US Pat. No. 6,737,054). In some embodiments, the cells from which human lung progenitor cells are derived are not modified using genetic means. Other approaches for positive selection include drug selection with concentration of the desired cells by density gradient centrifugation, described by Klug et al., Supra. Negative selection can be done by selecting and removing cells with undesirable markers or characteristics, such as fibroblast markers, epithelial cell markers, and the like.

  Undifferentiated ES cells express a gene that can be used as a marker for detecting the presence of undifferentiated cells. Polypeptide products of such genes can be used as negative selection markers. See, eg, US Patent Application No. 2003/0224411 Al; Bhattacharya (2004) Blood 103 (8): 2956-64; and Thomson (1998), each of which is incorporated herein by reference. Human ES cell lines include stage-specific embryonic antigen (SSEA) -3, SSEA-4, TRA-I-60, TRA-1-81, and alkaline phosphatase, but are undifferentiated It expresses cell surface markers that characterize non-human primate ES and human EC cells. Globo glycolipid GL7 having SSEA-4 epitope is formed by adding sialic acid to globo glycolipid Gb5 having SSEA-3 epitope. Thus, GL7 reacts with antibodies against both SSEA-3 and SSEA-4. Undifferentiated human ES cell lines do not stain for SSEA-1, but differentiated cells show strong staining for SSEA-1. Methods for growing undifferentiated hES cells are described in WO 99/20741, WO 01/51616, and WO 03/020920, the entire contents of which are hereby incorporated by reference.

  Exemplary cell surface markers expressed on lung progenitor cells include Sox2, Sox9, p63, FoxP2, ETV4 / 5, FoxA2, Nkx2.1, Gata6, ID2, CK5, NGFR, FoxJ1, CCSP, Scgb3a2, Muc5ac , T1a, Spc, and Scgn, but are not limited to these. Specific cell compositions having such cell surface marker combinations are exemplified herein in the Examples section.

In some embodiments, the human lung progenitor cells are an enriched cell population; that is, the percentage of human lung progenitor cells in the cell population (eg, the percentage of cells) is at least 10% of the total number of cells in the population. For example, the enriched population comprises at least 15% human lung progenitor cells, and at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the population , At least 95%, at least 99%, or even 100% contain human lung progenitor cells. In some embodiments, the cell population is at least 100 cells, at least 500 cells, at least 1000 cells, at least 1 × 10 ⁴ cells, at least 1 × 10 ⁵ cells, at least 1 × 10 ⁶ cells, at least 1 × 10 ⁷ cells. , At least 1 × 10 ⁸ , at least 1 × 10 ⁹ , at least 1 × 10 ¹⁰ , at least 1 × 10 ¹¹ , at least 1 × 10 ¹² , at least 1 × 10 ¹³ , at least 1 × 10 ¹⁴ , Contains at least 1 x 10 ¹⁵ or more cells.

  In one embodiment, the human lung progenitor cells described herein are not tumor cells or cancer cells. In such embodiments, human lung progenitor cells can be distinguished from tumor cells or cancer cells using, for example, a cell marker profile.

Scaffold compositions Biocompatible synthetic, natural, and semi-synthetic polymers can be used to synthesize polymer particles that can be used as scaffold materials. In general, with respect to the practice of the methods described herein, a scaffold may be used to allow lung progenitor cells to be isolated from the polymer prior to implantation, or the scaffold may degrade and remove over time in the subject. It is biodegradable so that it is not necessary. Thus, in one embodiment, the scaffold provides a temporary structure for human lung progenitor cells to grow and / or to deliver to a subject in need thereof. In some embodiments, the scaffold allows human lung progenitor cells to grow in a shape suitable for implantation or administration to a subject in need thereof, thereby removing the scaffold prior to implantation. Thus, the risk of rejection or allergic reaction initiated by the scaffold itself can be reduced.

  Examples of polymers that can be used include natural and synthetic polymers, with synthetic polymers being preferred in terms of reproducibility and sustained release kinetics. Synthetic polymers that can be used are poly (lactic acid) (PLA), poly (glycolic acid) (PGA), poly (lactide-co-glycolide) (PLGA), and other polyhydroxy acids, poly (caprolactone), polycarbonate Biodegradable polymers such as polyamides, polyanhydrides, polyphosphazenes, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates, and biodegradable polyurethanes; polyacrylic acid, ethylene-vinyl acetate polymers, and other acyls Non-biodegradable polymers such as substituted cellulose acetate and its derivatives; polyurethane, polystyrene, polyvinyl chloride, polyvinyl fluoride, poly (vinyl imidazole), chlorosulfonated polyolefins, and polyethylene oxide. Examples of biodegradable natural polymers include proteins such as albumin, collagen, fibrin, silk, synthetic polyamino acids, and prolamins; polysaccharides such as alginate and heparin; and other naturally occurring biodegradable sugar units These polymers are mentioned. Alternatively, a combination of the above polymers can be used.

  PLA, PGA, and PLA / PGA copolymers are particularly useful for forming biodegradable scaffolds. PLA polymers are usually prepared from cyclic esters of lactic acid. PLA polymers can be prepared using both L (+)-type and D (-)-type lactic acid, and an optically inactive DL-lactic acid mixture of D (-) and L (+) lactic acid. Methods for preparing polylactic acid are well reported in the patent literature. The following US patents, the teachings of which are incorporated herein by reference, describe in detail suitable polylactic acid, its properties and its preparation: US Patent No. 1,995,970 to Dorough; US Patent No. 2,703,316 to Schneider U.S. Pat. No. 2,758,987 to Salzberg; U.S. Pat. No. 2,951,828 to Zeile; U.S. Pat. No. 2,676,945 to Higgins; and U.S. Pat. No. 2,683,136 to Trehu;

  PGA is a homopolymer of glycolic acid (hydroxyacetic acid). In the conversion of glycolic acid to poly (glycolic acid), glycolic acid first reacts with itself to form a cyclic ester glycolide, and then in the presence of heat and catalyst to a high molecular weight linear polymer Is done. PGA polymers and their properties are described in more detail in Cyanamid Research Develops World's First Synthetic Absorbable Suture ", Chemistry and Industry, 905 (1970).

  The fibers can be formed by melt spinning, extrusion, casting, or other techniques well known in the polymer processing area. Preferred solvents are those that, when used to remove the scaffold prior to implantation, are either completely removed by processing or are biocompatible in the amount remaining after processing.

  The polymer used in the matrix should meet the mechanical and biochemical parameters necessary to provide a suitable support for subsequent growth and proliferation of the cells. Polymers are analyzed for mechanical properties such as tensile force using an Instron tester, for molecular weight of the polymer by gel permeation chromatography (GPC), for glass transition temperature by differential scanning calorimetry (DSC), and infrared (IR) spectroscopy Can be characterized with respect to the binding structure.

  The scaffold can be a scaffold of any desired shape and can include a wide range of geometric structures that are useful for the methods described herein. A non-limiting list of shapes includes, for example, hollow particles, tubes, sheets, cylinders, spheres, and fibers, among others. The shape or size of the scaffold should not substantially interfere with cell growth, cell differentiation, cell proliferation, or any other cellular process, or the scaffold may induce cell death, for example by apoptosis or necrosis Must not. In addition, the shape of the scaffold should be taken to ensure that adequate surface area is obtained for the nutrients from the surrounding medium to be delivered to the cells in the population so that cell viability is not compromised. is there. The porosity of the scaffold can also be varied as desired by one skilled in the art.

  In some embodiments, cell adhesion to the polymer comprises basement membrane components, agar, agarose, gelatin, gum arabic, type I, type II, type III, type IV, and type V collagen, fibronectin, laminin, glycosamino It is enhanced by coating the polymer with compounds such as glycans, polyvinyl alcohol, mixtures thereof, and other hydrophilic and peptide adhesive materials known to those skilled in the art of cell culture or tissue engineering. Examples of materials for coating polymer scaffolds include polyvinyl alcohol and collagen.

  In some embodiments, the scaffold can include decellularized lung tissue. Methods for producing decellularized lung tissue are known in the art, see eg WO2011 / 005306. Briefly, the decellularization process involves the step of chemically exfoliating the cells from lung tissue to remove cellular debris and leave an extracellular matrix structure. The human lung progenitor cells described herein can then be regenerated to the extracellular matrix, optionally with other bioactive agents. Such decellularized scaffolds can be prepared from a portion of the subject's own lungs, thus minimizing the risk of rejection or allergic reactions in response to regeneratively administered scaffolds.

  In some embodiments, it may be desirable to add bioactive molecules to the scaffold. A variety of bioactive molecules can be delivered using the matrices described herein. These are generally referred to herein as “factors” or “bioactive factors”.

  In one embodiment, the bioactive factor comprises a growth factor. Examples of growth factors include platelet-derived growth factor (PDGF), transforming growth factor α or β (TGFβ), bone morphogenetic protein 4 (BMP4), fibroblast growth factor 7 (FGF7), fibroblast growth factor 10 (FGF10), epidermal growth factor (EGF / TGFα), vascular endothelial growth factor (VEGF), some of which are also angiogenic factors.

  These factors are known to those skilled in the art and are commercially available or described in the literature. Bioactive molecules can be incorporated into the matrix and released over time by diffusion and / or degradation of the matrix, or they can be suspended with the cell suspension.

Treatment of Lung Disease / Disorders and Lung Injury The methods and compositions provided herein relate to the generation and use of human lung progenitor cells. Accordingly, provided herein are methods for treating and preventing lung injury or lung disease or disorder in a subject in need thereof. The methods described herein are for treating, ameliorating, preventing, or delaying a number of lung diseases or symptoms thereof, such as diseases that cause pathological damage to the structure of the lungs or airways and / or alveolar damage. Can be used. The terms “respiratory disorder”, “respiratory disorder”, “pulmonary disorder”, “pulmonary disorder”, “pulmonary disease” and “pulmonary disorder” are used interchangeably herein. Means any condition and / or disorder associated with the respiratory system and / or respiratory system, including the lung, thoracic cavity, bronchial, trachea, upper respiratory tract, airway, or other components or structures of the respiratory system.

  Such lung diseases include bronchoalveolar dysplasia (BPD), chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchodilation, pulmonary heart, pneumonia, lung abscess, acute bronchitis, chronic bronchitis, Emphysema, pneumonitis (eg, hypersensitivity pneumonitis or pneumonitis associated with radiation exposure), alveolar lung disease, and interstitial lung disease, environmental lung disease (eg, asbestos, smoke, or gas (Related to exposure), swallowing pneumonia, pulmonary hemorrhage syndrome, amyloidosis, connective tissue disease, systemic sclerosis, ankylosing spondylitis, pulmonary actinomycosis, alveolar proteinosis, lung anthrax, lung edema, lung embolism, lung Inflammation, pulmonary histocytosis X, pulmonary hypertension, surfactant deficiency, pulmonary neoplasia, pulmonary neoplasia, pulmonary nocardiosis, pulmonary tuberculosis, pulmonary vein occlusion disease, rheumatic lung disease, sarcoidosis, post-pneumonectomy, Wegener's granuloma Disease Allergic granulomatosis, granulomatous vasculitis, eosinophilia, asthma and airway hypersensitivity (AHR) (eg, mild intermittent asthma, mild persistent asthma, moderate persistent asthma, severe persistent) Asthma, acute asthma, chronic asthma, atopic asthma, allergic asthma or idiosyncratic asthma), allergic bronchopulmonary aspergillosis, chronic sinusitis, pancreatic insufficiency, lung or vascular inflammation, bacterial or viral infection, For example, Haemophilus influenzae, S. aureus, Pseudomonas aeruginosa infection or respiratory polynuclear virus (RSV) infection, or Grade I, II, III, or IV RDS Including acute or chronic adult or childhood respiratory distress syndrome (RDS), or RDS associated with, for example, sepsis, pneumonia, reperfusion, atelectasis, or chest trauma But it is not limited to these.

  Chronic obstructive pulmonary disease (COPD) includes diseases in which upper airway, middle airway, bronchiole, or airflow obstruction exists in the lung parenchyma, tracheal stenosis, tracheal right ventricular hypertrophic pulmonary hypertension, polychondritis, Bronchiectasis, bronchiolitis, such as idiopathic bronchiolitis, ciliary dyskinesia, asthma, emphysema, connective tissue disease, chronic bronchitis or bronchiolitis of lung transplants can be or can be related thereto.

The methods described herein also include airway epithelial damage, airway smooth muscle spasm, or airway hypersensitivity, airway mucosal edema, increased mucus secretion, excessive T cell activation, or desquamation, atelectasis, pulmonary heart , Pneumothorax, subcutaneous emphysema, dyspnea, cough, wheezing, shortness of breath, tachypnea, fatigue, decreased first-second forced expiratory volume (FEV ₁ ), arterial hypoxia, respiratory acidosis, IL-4, IL -5, undesired elevated levels of mediators such as IgE, histamine, substance P, neurokinin A, calcitonin gene related peptides or arachidonic acid metabolites such as thromboxane or leukotrienes (LTD ₄ or LTC ₄ ), and eosinophils, for example Treat acute or chronic lung diseases / disorders, or symptoms or complications thereof, including inflammation, including airway wall cell infiltration by lymphocytes, macrophages or granulocytes It can be used to improve.

  Any of these and other respiratory or pulmonary conditions or symptoms are known in the art. See, for example, The Merck Manual, 17th edition, MH Beers and R. Berkow editors, 1999, Merck Research Laboratories, Whitehouse Station, NJ, ISBN 0911910-10-7, or other references cited herein. I want.

  As used herein, the terms “administering”, “introducing”, and “transplanting” refer to cells, eg, lung progenitor cells described herein, so that a desired effect is obtained. Used interchangeably in the context of placing in a subject by a method or route that results in at least partial localization of the introduced cells at the desired site, such as a damaged or repair site. Cells, such as lung progenitor cells or differentiated progeny thereof (eg, airway progenitor cells, basal cells, Clara cells, ciliated cells or goblet cells) can be implanted directly into the respiratory airway or of implanted cells or cellular components It can be administered by any suitable route that results in delivery to a desired location in a subject that remains at least partially alive. Cell survival after administration to a subject can be a few hours, eg, as short as 24 hours to a few days, as long as several years, ie, long-term engraftment. For example, in some embodiments of the aspects described herein, an effective amount of lung progenitor cells is administered directly to the lungs of an infant with bronchopulmonary dysplasia by intratracheal administration. In other embodiments, pulmonary progenitor cells can be administered by an indirect systemic route of administration, such as an intraperitoneal or intravenous route.

  When provided prophylactically, the lung progenitor cells described herein can be administered to a subject before any symptoms of a lung disorder, such as an asthma attack, or to a premature infant. Accordingly, prophylactic administration of a lung progenitor cell population is useful for preventing lung disorders disclosed herein.

  When provided therapeutically, pulmonary progenitor cells are provided at the onset (or post-occurrence) of a pulmonary disorder symptom or indicator, eg, at the onset of COPD.

  In some embodiments of the aspects described herein, the lung progenitor cell population administered according to the methods described herein comprises allogeneic lung progenitor cells obtained from one or more donors. Including. As used herein, “allogeneic” refers to lung progenitor cells or lung progenitor cells obtained from one or more different donors of the same species that do not have the same gene at one or more loci. Means a biological sample containing. For example, a lung progenitor cell population administered to a subject can be derived from umbilical cord blood obtained from one or more unrelated donor subjects or from one or more non-identical siblings . In some embodiments, syngeneic lung progenitor cell populations can be used, such as cells obtained from genetically identical animals or cells obtained from genetically identical twins. In other embodiments of this aspect, the lung progenitor cells are autologous cells. That is, lung progenitor cells are obtained or isolated from a subject and administered to the same subject, i.e. the donor and recipient are the same.

  Depending on the disease / disorder or injury being treated, and depending on the location of the lung injury, either undifferentiated human lung progenitor cells or differentiated cells thereof can be administered to the subject.

Pharmaceutically Acceptable Carriers The methods described herein for administering human lung progenitor cells to a subject involve using a therapeutic composition comprising lung progenitor cells. The therapeutic composition comprises a physiologically acceptable carrier with the cell composition and optionally at least one additional bioactive agent described herein which is dissolved or dispersed therein as an active ingredient. including. In preferred embodiments, the therapeutic composition is substantially non-immunogenic when administered to a mammalian or human patient for therapeutic purposes, unless it is desired. As used herein, “pharmaceutically acceptable”, “physiologically acceptable”, and grammatical variations thereof are interchangeable when they refer to compositions, carriers, diluents, and reagents. Used to indicate that the material can be administered to a mammal without causing undesirable physiological effects such as nausea, dizziness, upset stomach, transplant rejection, allergic reactions and others. A pharmaceutically acceptable carrier does not promote the generation of an immune response to the substance with which it is mixed, unless it is desirable to do so. The preparation of a composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable preparations, either in liquid solution or suspension, but are in solid dosage forms suitable to be in solution or suspension in liquid prior to use. Can be prepared similarly.

  In general, the human lung progenitor cells described herein are administered as a suspension with a pharmaceutically acceptable carrier. One skilled in the art will recognize that the pharmaceutically acceptable carrier used in the cell composition is in an amount of buffer, compound, cryoprotectant, preservative, or other that substantially interferes with the viability of the cells delivered to the subject. You will recognize that it contains no substances. Formulations containing cells can include, for example, osmotic buffers that can maintain cell membrane integrity, and optionally nutrients to maintain cell viability or enhance engraftment upon administration. Such formulations and suspensions are known to those skilled in the art and / or can be adapted for use with human lung progenitor cells described herein using routine experimentation.

  The cell composition can also be emulsified or presented as a liposomal composition so long as the emulsification technique does not adversely affect cell viability. Cells and any other active ingredients can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredients in amounts suitable for use in the therapeutic methods described herein. .

  Additional materials included in the cell compositions described herein can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts are, for example, acid addition salts formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid and others (depending on the free amino group of the polypeptide). Formed). Salts formed by free carboxyl groups also include inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, as well as isopropylamine, trimethylamine, 2-ethylaminoethanol Can be derived from organic bases such as histidine, procaine, and others. Physiologically acceptable carriers are well known in the art. Exemplary liquid carriers include buffers other than the active ingredient and water, and materials such as sodium phosphate, physiological saline, or both, such as phosphate buffered saline, at physiological pH values. A sterilized aqueous solution containing Still further, the aqueous carrier may contain more than one buffer salt and salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. The liquid composition may also include a liquid phase in addition to and excluding water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active compound used in the cell compositions described herein that is effective to treat a particular disorder or condition will be determined by standard clinical techniques, depending on the nature of the disorder or condition. Can be done.

Administration and Efficacy Provided herein is a method of treating a lung disease, lung disorder, or lung injury comprising administering human lung progenitor cells or differentiated progeny thereof to a subject in need thereof.

  Parameters that are measured or measurable include clinically detectable markers of disease, such as elevated or suppressed levels of clinical or biological markers, as well as clinically acceptable symptoms or markers of disease or disorder. Parameters related to the scale. However, it is understood that the total daily dosage of the compositions and formulations disclosed herein will be determined by the attending physician within the scope of sound medical judgment. The exact amount required will vary depending on factors such as the type of disease being treated.

  As used herein, the term `` effective amount '' means the amount of human lung progenitor cell population or progeny thereof that is required to reduce lung injury or at least one or more symptoms of lung disease or disorder. , Related to a sufficient amount of the composition to provide the desired effect, eg, to treat a subject with smoking-induced injury or cystic fibrosis. Thus, the term “therapeutically effective amount” is sufficient to promote a particular effect when administered to a typical subject, such as a subject with or at risk for lung disease or disorder. Means an amount of a human lung progenitor cell or a composition comprising human lung progenitor cells. An effective amount as used herein also alters the course of disease symptoms, preventing or delaying the onset of disease symptoms (eg, but not limited to slowing the progression of disease symptoms). Or an amount sufficient to reverse the symptoms of the disease. For any given case, one of ordinary skill in the art will understand that an appropriate “effective amount” can be determined using routine experimentation.

  In some embodiments, the subject is first diagnosed with a disease or disorder that affects lung tissue prior to administering the cells according to the methods described herein. In some embodiments, the subject is first diagnosed with having a lung disease or disorder or at risk of developing a lung disease or disorder prior to administering the cells. For example, premature babies can have a significant risk of developing lung disease or disorder.

For use in the various aspects described herein, an effective amount of human lung progenitor cells is at least 10 ² lung progenitor cells, at least 5 × 10 ² lung progenitor cells, at least 10 ³ lung progenitor cells, At least 5 × 10 ³ lung precursor cells, at least 10 ⁴ lung precursor cells, at least 5 × 10 ⁴ lung precursor cells, at least 10 ⁵ lung precursor cells, at least 2 × 10 ⁵ lung precursor cells, at least lung precursor cells 3 × 10 ⁵ , lung progenitor cells at least 4 × 10 ⁵ , lung progenitor cells at least 5 × 10 ⁵ , lung progenitor cells at least 6 × 10 ⁵ , lung progenitor cells at least 7 × 10 ⁵ , lung progenitor cells at least 8 × 10 ⁵ , lung progenitor cells at least 9 × 10 ⁵ , lung progenitor cells at least 1 × 10 ⁶ , lung progenitor cells at least 2 × 10 ⁶ , lung progenitor cells at least 3 × 10 ⁶ , lung progenitor cells at least 4 × 10 ⁶ cells, lung progenitor cells at least 5 × 10 ⁶ cells, lung progenitor cells reduced Also 6 × 10 ⁶ cells, including lung progenitor cells at least 7 × 10 ⁶ cells, lung progenitor cells at least 8 × 10 ⁶ cells, lung progenitor cells at least 9 × 10 ⁶ cells, or multiples thereof. Lung progenitor cells can be derived from one or more donors or can be obtained from autologous origin. In some embodiments of the aspects described herein, lung progenitor cells are grown in culture prior to administration to a subject in need thereof.

  Exemplary modes of administration for use in the methods described herein include injection, intrapulmonary (including intranasal and intratracheal) infusion, inhalation as an aerosol (including intranasal), and implantation (scaffolding). With or without material), but is not limited thereto. “Injection” includes, but is not limited to, intravenous, intramuscular, intraarterial, intradermal, intraperitoneal, transtracheal, and subcutaneous. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than intestinal and surface administration, usually by injection, and are intravenous, intraperitoneal, intramuscular. Including, but not limited to, intraarterial, intradermal, transtracheal, and subcutaneous administration.

  In some embodiments, the therapeutically effective amount of lung progenitor cells is administered using pulmonary administration, such as intranasal or intratracheal route. In some aspects of these methods, a therapeutically effective amount of lung progenitor cells is administered using systemic administration, such as intraperitoneal or intravenous routes. In other aspects of these methods, a therapeutically effective amount of lung progenitor cells is administered using both intrapulmonary and intraperitoneal administration. These methods are particularly intended for therapeutic or prophylactic treatment of human subjects having or at risk of having a lung disease or disorder. The human lung progenitor cells described herein can be administered to a subject with any lung disease or disorder by any suitable route that provides effective treatment in the subject. In some embodiments of the aspects described herein, subjects with a pulmonary disorder are first selected prior to administration of the cells.

  In some embodiments, an effective amount of lung progenitor cells is administered to the subject by intrapulmonary administration or delivery. “Intrapulmonary” administration or delivery as defined herein includes, but is not limited to, lung progenitor cell populations such as Nkx2.1 + Sox2 + lung progenitor cells, transtracheal, intratracheal, and intranasal administration It does not mean that all routes of administration are administered in such a way that direct contact between the subject's airways and these cells occurs. In some such embodiments, the cells are injected into the nasal cavity or trachea. In some embodiments, the cells are inhaled directly by the subject. In some embodiments, intrapulmonary delivery of cells includes a method of administration in which the cells are administered to the intubated subject, eg, as a cell suspension, via a tube placed in the trachea or “tracheal intubation”.

  As used herein, “tracheal intubation” means placing a flexible tube, such as a plastic tube, in the trachea. The most common tracheal intubation is referred to herein as “oral endotracheal intubation”, with the help of a laryngoscope, passing the endotracheal tube through the mouth, pharynx, and vocal cords to the trachea. is there. The sphere is then inflated near the distal end of the tube to help secure the tube in place and to protect the airways from blood, vomit, and secretions. In some embodiments, the cells are administered to a subject having “nasal endotracheal intubation” defined as tracheal intubation that passes the tube through the nose, pharynx, vocal cords, and trachea.

  In some embodiments, an effective amount of lung progenitor cells is administered to a subject by systemic administration, such as intravenous administration.

  As used herein, the phrases “systemic administration”, “administered systemically”, “peripheral administration”, and “administered peripherally” refer to cells entering the subject's circulatory system, thus metabolism and Meaning that the lung progenitor cell population is administered other than directly to a target site, tissue, or organ, such as the lung, to undergo other similar processes.

  In some embodiments of the aspects described herein, one or more routes of administration are used in the subject to achieve a distinct effect. For example, lung progenitor cells can be administered to a subject by both intratracheal and intraperitoneal routes of administration, respectively, to treat or repair lung epithelium and to repair and regenerate pulmonary blood vessels. In such embodiments, different effective amounts of isolated or enriched lung progenitor cells can be used for each route of administration.

  When using aerosol administration, the nebulizer device requires a formulation suitable for dispensing a particular composition. The choice of formulation depends on the specific composition used and the number of lung progenitor cells administered, and such formulation can be adjusted by one skilled in the art. However, by way of example, when the composition is lung progenitor cells in a pharmaceutically acceptable carrier, the composition is in an appropriate buffer (eg, saline buffer) at an effective concentration of cells per mL of solution. It can be a suspension of cells in it. The formulation can also include cellular nutrients, simple sugars (eg, to regulate osmotic pressure) or other components to maintain cell survival.

  Typically, each formulation for aerosol delivery by nebulizer is specific to the type of device used and is suitable propulsion in addition to the usual diluents, adjuvants and / or carriers useful in therapy. It can involve the use of materials.

  In some embodiments, additional substances that aid in the treatment of the subject can be administered before or after treatment with lung progenitor cells as described herein. Such additional materials can be used to prepare lung tissue for administering progenitor cells. Alternatively, additional substances can be administered after lung progenitor cells to support engraftment and growth of the administered cells in the damaged lung. Such additional materials can generally be formulated for use with a fixed dose inhaler device comprising fines containing proteins or small molecules suspended in a propellant with the aid of a surfactant. Propellants include trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, or hydrocarbons Of any conventional material used for this purpose. Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid may also be useful as a surfactant.

  Formulations for dispensing from powder inhaler devices include finely divided powders containing proteins or small molecules, as well as bulking agents such as lactose, sorbitol, sucrose, or mannitol in amounts that promote dispersion of the powder from the device. For example in an amount of 50 to 90% by weight of the formulation. The protein material should be conveniently prepared in a particulate form with an average particle size of less than 10 μm (or microns), most preferably 0.5 to 5 μm, in order to be most effectively delivered to the distal lung.

  Nasal delivery of proteins or other substances in addition to lung progenitor cells or their progeny is similarly contemplated. With nasal delivery, proteins or other substances enter the bloodstream directly after administration of the therapeutic product to the nose, without the need to deposit the product in the lungs. Formulations for nasal delivery include formulations with dextran or cyclodextran.

  The efficacy of the treatment can be determined by a clinician of ordinary skill. However, any one or all of the symptoms of pulmonary disease, lung injury, and / or pulmonary disorder, or other clinically observed symptom or marker comprise human lung progenitor cells as described herein A treatment is considered to be an “effective treatment” as the term is used herein if it is reduced by, for example, at least 10% after treatment with an object. Methods for measuring these indicators are known to those skilled in the art and / or are described herein.

  Indicators of pulmonary disease or disorder or injury include functional indicators, such as measurements of lung volume and lung function, and oxygen saturation (eg, tissue oxygen saturation or systemic arterial oxygen saturation) and biochemical indicators.

  With respect to idiopathic pulmonary fibrosis, for example, symptom improvement includes at least a 10% increase in predicted forced vital capacity (FVC) compared to pre-treatment values. FVC is the total volume of air exhausted after a sufficient intake. Patients with obstructive pulmonary disease usually have normal or negligible decreased vital capacity. Patients with restrictive lung disease have reduced vital capacity.

  Another measure is FEV1 (1 second forced vital capacity). This is the volume of air discharged during the first second during the maximum expiratory effort. FEV1 is reduced in both obstructive and restrictive lung disease. FEV1 decreases due to increased airway resistance in obstructive pulmonary disease. FEV1 decreases due to low vital capacity in restrictive lung disease.

  The relevant measurement is FEV1 / FVC. This is the percentage of vital capacity excreted in the first second of exhaled breath. In healthy patients, FEV1 / FVC is usually about 70%. In patients with obstructive pulmonary disease, FEV1 / FVC decreases and can be as low as 20-30% in severe obstructive airway disease. Restraint disorder has almost normal FEV1 / FVC.

  If necessary or desired, an animal model of lung injury or disease can be used to measure the effectiveness of a particular composition described herein. As an example, a bleomycin-induced lung injury model of acute lung injury (ALI) can be used. Animal models of pulmonary function are useful for monitoring bronchoconstriction, allergic reactions, delayed airway hyperresponsiveness in response to inhaled allergens, among other endpoints, such as head-out or body plethysmography Models (see, eg, Hoymann, HG et al., J Pharmacol Toxicol Methods (2007) 55 (1): 16-26). Exemplary animal models of asthma, including allergic asthma (eg, acute and chronic allergic asthma) models are known in the art. Nials and Uddin. (2008) Dis Model Mech 1: 213-220; Zosky and Sly (2007) Clin Exp Allergy 37 (7): 973-88; and Kumar and Foster. (2002) Am J Respir Cell Mol Biol 27 (3): Please refer to 267-72. Animal models of pneumonia have been reviewed by Mizgerd and Skerrett (2008) Am J Physiol Lung Cell Mol Physiol 294: L387-L398. In addition, small animal imaging can be applied to lung pathophysiology (Brown RH, et al., Proc Am Thorac Soc (2008) 5: 591-600).

Screening Assays The compositions described herein are useful for screening agents for inducing differentiation of human lung progenitor cells or treating lung diseases or disorders.

  In some embodiments, methods, assays, systems, and methods for developing specific in vitro assays of isolated human lung progenitor cells or isolated human disease-specific lung cells derived from such human lung progenitor cells It can be used in a kit. Such assays for drug screening and toxicity testing are that they are cells of human origin, do not require immortalization of cell lines, and do not require tissues from cadaver that do not reflect well the normal human cell physiology Have advantages over existing assays. For example, the methods, assays, systems, and kits described herein can be used to identify and / or test substances that can promote differentiation along the lung lineage. Additionally or alternatively, the methods, assays, systems, and kits are used to identify and / or test substances that are useful for treating lung diseases or disorders, or for preventing / treating lung injury. sell.

  Thus, herein, (a) contacting an isolated human lung progenitor cell described herein or its progeny with a test compound, and (b) determining any effect of the compound on the cell. A method of screening a test compound for biological activity is provided. In one embodiment, the screening method further comprises generating human lung progenitor cells or human lung disease specific cells as disclosed herein. In one embodiment, lung progenitor cells are first differentiated into the desired lung cell phenotype. The effect on the cell can be an effect that can be observed directly or indirectly by using a reporter molecule.

  As used herein, the term “biological activity” or “biological activity” refers to the ability of a test compound to affect a biological sample. Biological activity can include, but is not limited to, stimulation, inhibition, modulation, toxicity, or induction of a lethal response in a biological assay. For example, biological activity is the ability of a compound to modulate the effect of an enzyme, the ability to block a receptor, the ability to stimulate a receptor, the ability to modulate the expression level of one or more genes, cell proliferation It can mean the ability to regulate, the ability to regulate cell division, the ability to regulate cell metabolism, the ability to regulate differentiation, the ability to regulate cell morphology, or a combination thereof. In some examples, biological activity can refer to the ability of a test compound to produce a toxic effect in a biological sample.

  As discussed above, a specific lineage can be a lineage that is a phenotype and / or genotype of a disease (eg, lung disease). Alternatively, the specific lineage may be a lineage that is a phenotype and / or genotype of an organ and / or tissue, or part thereof (eg, lung).

  As used herein, the term “test compound” or “candidate substance” refers to a substance or collection of substances (eg, a compound) that is screened for whether it can have an effect on a cell. Test compounds are chemical compounds, mixtures of chemical compounds, such as polysaccharides, small organic or inorganic molecules (eg, molecules having a molecular weight of less than 2000 daltons, less than 1000 daltons, less than 1500 daltons, less than 1000 daltons, or less than 500 daltons) Made from biological materials such as biopolymers such as peptides, proteins, peptide analogs and analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, bacteria, plants, fungi, or animal cells or tissues Can include a wide variety of different compounds, including extracted extracts, naturally occurring or synthetic compositions.

  Depending on the particular embodiment practiced, the test compound can be provided free in solution or can be bound to a carrier or solid support, such as a bead. A number of suitable solid supports can be used to immobilize the test compound. Examples of suitable solid supports include agarose, cellulose, dextran (ie, commercially available as Sephadex, Sepharose), carboxymethylcellulose, polystyrene, polyethylene glycol (PEG), filter paper, nitrocellulose, ion exchange resin, plastic film. , Polyamine methyl vinyl ether maleic acid copolymer, glass beads, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk and the like. In addition, for the methods described herein, test compounds can be screened individually or in groups. Group screening is particularly useful when the effective test compound hit rate is expected to be so low that no more than one positive result can be expected for a given group.

  A number of small molecule libraries are known in the art and are commercially available. These small molecule libraries can be screened using the screening methods described herein. A chemical library or compound library is a collection of stored chemicals that can be used with the methods described herein to screen candidate substances for a particular effect. The chemical library contains information regarding the chemical structure, purity, quantity, and physicochemical characteristics of each compound. Compound libraries include, for example, Enzo Life Sciences (TM), Aurora Fine Chemicals (TM), Exclusive Chemistry Ltd. (TM), ChemDiv, ChemBridge (TM), TimTec Inc. (TM), AsisChem (TM), and Princeton It can be obtained by purchasing from Biomolecular Research ™.

  The compound may be tested at any concentration that can exert an effect on the cells relative to the control for an appropriate period of time, but is not limited to this. In some embodiments, the compound is at a concentration ranging from about 0.01 nM to about 100 mM, from about 0.1 nM to about 500 μM, from about 0.1 μM to about 20 μM, from about 0.1 μM to about 10 μM, or from about 0.1 μM to about 5 μM. To be tested.

  Compound screening assays can be used in high throughput screening. High throughput screening is the process of testing a library of compounds for a given activity. High throughput screening seeks to screen large numbers of compounds quickly and simultaneously. For example, using a microtiter plate and automated assay equipment, a laboratory can perform as many as 100,000 assays a day simultaneously.

  The compound screening assays described herein include more than one measurement of cellular or reporter function (eg, measurement of more than one parameter and / or one or more at multiple points in the course of the assay). Parameter measurement). By measuring multiple times, biological activity can be followed over the period of incubation with the test compound. In one embodiment, reporter function is measured at multiple time points so that the effect of the test compound can be monitored at different incubation periods.

  The screening assay can be followed by an assay that further identifies whether the identified test compound has desirable properties for the intended use. For example, a screening assay can be followed by a second assay selected from the group consisting of either bioavailability, toxicity, or pharmacokinetic measurements, but is not limited to these methods. Absent.

Kits Another aspect of the technology described herein is a kit for treating a pulmonary disease or disorder, a kit for screening candidate substances, and / or differentiating human stem cells into human lung progenitor cells. And a kit for differentiating human lung progenitor cells into specific types or types of human lung cells. Described herein are kit components that can be included in one or more of the kits described herein.

  In one embodiment, the kit described herein can include human lung progenitor cells, the term of which is used herein. In one embodiment, one or more signaling pathway agonists or antagonists that promote stem cell differentiation are included in the kit. In another embodiment, stem cells using one or more TGF-β receptor inhibitors, one or more BMP agonists, one or more FGF agonists and methods described herein, for example. The components described herein, such as instructions for converting (eg, embryonic stem cells, isolated pluripotent stem cells, anterior foregut endoderm cells, or definitive endoderm cells) to human lung progenitor cells, Included in kit.

  Another aspect of the technology disclosed herein relates to a kit for producing human lung progenitor cells according to the methods disclosed herein. In some embodiments, the components described herein can be provided in any combination as a single component or as a kit. The kit includes a composition comprising the components described herein, eg, a compound described herein, eg, a compound for differentiating human stem cells into lung progenitor cells, or a cocktail of compounds or reagents. Such a kit may optionally include one or more substances capable of detecting lung progenitor cell markers or lung cell markers or sets thereof. In addition, the kit optionally includes informational material.

  In some embodiments, the compounds in the kit may in some embodiments be provided in a water resistant or airtight container that is substantially free of other components of the kit. For example, a signaling pathway or differentiation pathway modulating compound is supplied in more than one container, for example, a container with sufficient reagents for a predetermined number of differentiation reactions, such as one, two, three or more containers Can be done. The container or containers described herein can be provided in any dosage form, eg, in liquid, dry, or lyophilized form. It is preferred that the compounds described herein are substantially pure and / or sterile. When one or more signal transduction pathway modulating compounds described herein are provided in a liquid solution, the liquid solution is preferably an aqueous solution, preferably a sterile aqueous solution. When the compounds described herein are provided in dry form, reconstitution is generally performed by the addition of a suitable solvent. Solvents such as sterile water or buffers can optionally be provided in the kit.

  The informational material can be instructions, instructions, sales, or other materials related to the methods described herein and / or the use of the compounds described herein with respect to the methods described herein. . The information material of the kit is not limited to its shape. In one embodiment, the informational material can include information regarding the production of the compound, the molecular weight of the compound, concentration, expiration date, batch or manufacturing location information, and so forth. In one embodiment, the informational material relates to a method of using or administering the compound.

  In one embodiment, the informational material is in a manner suitable for performing treatment of lung injury or lung disease or disorder, such as a suitable dose, dosage form, or mode of administration (eg, as described herein). Instructions for administering human lung progenitor cells described herein in a dose, dosage form, or mode of administration. In another embodiment, the informational material can include instructions for differentiating human embryonic stem cells into human lung progenitor cells. Alternatively, the informational material can include instructions for screening candidate substances for treating lung disease or disorder.

  In addition to the compounds described herein, the composition of the kit may comprise a solvent or buffer, a stabilizer, a preservative, and / or for example to differentiate stem cells (eg, in vitro) or herein. Other ingredients may be included such as additional substances for treating the conditions or disorders described in. Alternatively, other components different from the cells or signaling pathways or differentiation pathway modulating compounds described herein can be included in the kit, but can be included in different compositions or containers. In such embodiments, the kit may include instructions for mixing the compounds described herein and other components, or instructions for using the compounds described herein with other components, such as It may contain instructions for mixing the two substances before use or administration.

  The kit may contain components for detecting markers such as human lung progenitor cells, ES cells, iPS cells, thyroid lineage cells, neural lineage cells and the like. In addition, the kit can include one or more antibodies that bind to cell markers, or primers for RT-PCR or PCR reactions, such as semi-quantitative or quantitative RT-PCR or PCR reactions. Such components can be used to assess activation of lung cell specific markers or loss of ES cells, iPSCs, thyroid lineage or neural lineage markers. Where the detection reagent is an antibody, the antibody can be supplied in a dry preparation, eg, lyophilized, or in solution. The antibody or other detection reagent can be linked to a label for use in detection, such as a radiolabel, a fluorescent label (eg, GFP) or a colorimetric label. Where the detection reagent is a primer, it can be supplied in a dry preparation, such as lyophilized or in solution.

  The kit may also include one or more reagents to enhance the production efficiency of induced pluripotent stem cells, such as an HDAC inhibitor (eg, valproic acid) or a DNA methyltransferase inhibitor (eg, 5 AzaC) .

  In one embodiment, the kit comprises a cell or tissue medium for definitive endoderm production. In one embodiment, the medium comprises B27, activin A and ZSTK474. An exemplary definitive endoderm production medium contains 2% B27, 20 ng / mL activin A, and 0.2-0.5 μM ZSTK474.

  The kit is typically provided with its various elements contained in one package, eg, fiber material, eg, cardboard, or polymer, eg, a styrofoam box. The encapsulation is configured to maintain an internal and external temperature difference, for example, it may provide an insulating property for maintaining the reagent at a preselected temperature for a preselected period.

The present invention may be as defined in any one of the following numbered paragraphs.
1. Isolated human Nkx2.1 positive Sox2 positive proximal airway multipotent progenitor cells.
2. The isolated cell of paragraph 1, which is Tuj1 negative and Pax8 negative.
3. The isolated cell according to paragraph 1, which is proliferative and differentiates into airway basal stem cells, ciliated cells, Clara cells, neuroendocrine cells, or squamous epithelial cells under selected differentiation conditions.
4. Isolated human Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells.
5. The isolated cell of paragraph 4, which is FoxP2 positive and / or ID2 positive.
6. The isolated cell of paragraph 4, which is proliferative and differentiates into any epithelial lung cell when placed in selected differentiation conditions.
7. Airway basal stem cells, ciliated cells, Clara cells, mucin-secreting goblet cells, type I alveolar cells, type II alveolar cells, squamous epithelial cells, bronchoalveolar stem cells, bronchopulmonary when placed in selected differentiation conditions 7. The isolated cell of paragraph 6, which differentiates into a tubal junction stem cell, a migratory CK14 + cell, or a neuroendocrine cell.
8. Isolated human Nkx2.1 positive p63 positive multipotent airway basal stem cells.
9. The isolated cell of paragraph 8, which is proliferative and differentiates into ciliated cells, Clara cells, mucin-secreting goblet cells, or basal cells when subjected to selected differentiation conditions.
10. The isolated cell of paragraph 8, wherein the multipotent airway basal stem cell does not express a Clara cell marker, ciliary cell marker, neuroendocrine cell marker, or squamous cell marker.
11. The isolated cell of paragraph 8, which is Sox2-positive.
12. The isolated cell of paragraph 8, which is CK5 positive and / or NGFR positive.
13. The isolated cell of paragraph 1, 5, or 8, which is a disease specific cell.
14. A composition comprising isolated human Nkx2.1-positive Sox2-positive proximal airway multipotent progenitor cells and a scaffold.
15. The composition of paragraph 14, wherein the scaffold is implantable in the subject.
16. The composition of paragraph 14, wherein the cell is autologous to the subject into which the composition is to be implanted.
17. The composition according to paragraph 14, wherein the scaffold is biodegradable.
18. The composition of paragraph 14, wherein the scaffold comprises natural fibers, synthetic fibers, decellularized lung tissue, or combinations thereof.
19. The composition according to paragraph 18, wherein the natural fiber is selected from the group consisting of collagen, fibrin, silk, thrombin, chitosan, chitin, alginic acid, hyaluronic acid, and gelatin.
20. Synthetic fiber is polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactide-co-glycolide) (PLGA), poly (caprolactone), diol / diacid type aliphatic polyester, polyester -Amide / polyester-urethane, poly (valerolactone), poly (hydroxylbutyric acid), polybutylene terephthalate (PBT), polyhydroxyhexanoic acid (PHH), polybutylene succinic acid (PBS), and poly (hydroxyl valeric acid) 20. The composition according to paragraph 19, wherein the composition is selected from the group consisting of a representative biodegradable aliphatic polyester.
21. The composition of paragraph 14, wherein the proximal airway multipotent progenitor cells are Tuj1 negative and / or Pax8 negative.
22. A composition comprising isolated human Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells and a scaffold.
23. The composition according to paragraph 22, wherein the multipotent lung progenitor cells are FoxP2 positive and / or ID2 positive.
24. The composition of paragraph 22, wherein the scaffold is implantable in the subject.
25. The composition according to paragraph 22, wherein the cells are autologous to the subject into which the composition is to be implanted.
26. The composition of paragraph 22, wherein the scaffold is biodegradable.
27. The composition of paragraph 22, wherein the scaffold comprises natural fiber, synthetic fiber, decellularized lung, or a combination thereof.
28. The composition according to paragraph 27, wherein the natural fiber is selected from the group consisting of collagen, fibrin, silk, thrombin, chitosan, chitin, alginic acid, hyaluronic acid, and gelatin.
29. Synthetic fibers are polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactide-co-glycolide) (PLGA), poly (caprolactone), diol / diacid type aliphatic polyester, polyester -Amide / polyester-urethane, poly (valerolactone), poly (hydroxylbutyric acid), polybutylene terephthalate (PBT), polyhydroxyhexanoic acid (PHH), polybutylene succinic acid (PBS), and poly (hydroxyl valeric acid) 29. The composition of paragraph 28, wherein the composition is selected from the group consisting of a representative biodegradable aliphatic polyester.
30. A composition comprising isolated human Nkx2.1 positive p63 positive multipotent airway basal stem cells and a scaffold.
31. The composition according to paragraph 30, wherein the airway basal stem cells are CK5 positive and / or NGFR positive.
32. The composition according to paragraph 30, wherein the scaffold is implantable in the subject.
33. The composition of paragraph 32, wherein the cell is autologous to the subject into which the composition is to be implanted.
34. The composition according to paragraph 30, wherein the scaffold is biodegradable.
35. The composition of paragraph 30, wherein the scaffold comprises natural fibers, synthetic fibers, decellularized lung tissue, or combinations thereof.
36. The composition according to paragraph 35, wherein the natural fiber is selected from the group consisting of collagen, fibrin, silk, thrombin, chitosan, chitin, alginic acid, hyaluronic acid, and gelatin.
37. Synthetic fibers are polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactide-co-glycolide) (PLGA), poly (caprolactone), diol / diacid type aliphatic polyester, polyester -Amide / polyester-urethane, poly (valerolactone), poly (hydroxylbutyric acid), polybutylene terephthalate (PBT), polyhydroxyhexanoic acid (PHH), polybutylene succinic acid (PBS), and poly (hydroxyl valeric acid) 37. The composition according to paragraph 36, selected from the group consisting of a representative biodegradable aliphatic polyester.
38. The composition according to paragraphs 14, 22, or 30, wherein the cell is a disease specific cell.
39. Human foregut endoderm cells with FGF2, WNT, and BMP4, each for a period and at a concentration sufficient to differentiate human foregut endoderm cells into Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells. A method of producing a human lung progenitor cell or human lung progenitor cell population that is Nkx2.1 positive, Tuj1 negative, and Pax8 negative, comprising a step of contacting.
40. The method of paragraph 39, wherein the contacting step occurs for at least 2 days.
41. Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells, BMP7, FGF7, WNT antagonist, and MAPKK / ERK antagonist, Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells, Nkx2.1 positive Sox2 positive nearby 40. The method of paragraph 39, further comprising contacting for a period and at a concentration sufficient to differentiate into peripheral airway multipotent progenitor cells.
42. The method of paragraph 41, wherein the Wnt antagonist comprises IWR-1.
43. The method of paragraph 41, wherein the MAPKK / ERK antagonist comprises PD98059.
44. The method of paragraph 41, wherein the contacting step occurs for at least 4 days.
45. Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells with BMP7, FGF7, WNT antagonist, and MAPKK / ERK antagonist, Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells each with Nkx2.1 positive Sox9 positive 42. The method of paragraph 41, further comprising the step of contacting for a period and at a concentration sufficient to differentiate into coordinate multipotent lung progenitor cells.
46. The method of paragraph 45, wherein the Wnt antagonist comprises IWR-1.
47. The method of paragraph 45, wherein the MAPKK / ERK antagonist comprises PD98059.
48. The method of paragraph 45, wherein the contacting step occurs for at least 4 days.
49. The method of paragraph 39, wherein the culture of foregut endoderm cells is derived from embryonic stem (ES) cells or induced pluripotent stem cells (iPSC).
50. Culture of Nkx2.1 positive Tuj1 negative Pax8 negative cells with B27, BMP7, FGF7, and WNT antagonist, Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells each with Nkx2.1 positive p63 positive multipotent airways 40. The method of paragraph 39, further comprising the step of contacting for a period and concentration sufficient to differentiate into basal stem cells.
51. The method of paragraph 39, further comprising contacting Noggin with a culture of Nkx2.1 positive Tuj1 negative Pax8 negative cells.
52. The method of paragraph 50, wherein the contacting step occurs for at least 10 days.
53. A lung disease or disorder or lung in a subject comprising administering a composition comprising isolated human lung progenitor cells and a pharmaceutically acceptable carrier to a subject having a lung disease or disorder or lung injury How to treat the damage.
54. Isolated human lung progenitor cells are Nkx2.1 positive Sox2 positive proximal airway multipotent progenitors, Nkx2.1 positive Sox9 positive distal multipotent progenitor cells, Nkx2.1 positive p63 positive multipotent airways 54. The method of paragraph 53, wherein the method is selected from the group consisting of basal stem cells or differentiated cells thereof.
55. The method of paragraph 53, wherein the proximal airway multipotent progenitor cells are Tuj1 negative and / or Pax8 negative.
56. The method of paragraph 53, wherein the distal multipotent lung progenitor cells are FoxP2 and / or ID2 positive.
57. The method of paragraph 53, wherein the airway basal stem cells are CK5 positive and / or NGFR positive.
58. The method of paragraph 53, wherein the composition is administered to the lung.
59. The method of paragraph 53, wherein the isolated human lung progenitor cells are autologous to the subject to which the composition is administered.
60. The method of paragraph 53, wherein the composition further comprises a scaffold.
61. The method of paragraph 60, wherein the scaffold is implantable in the subject.
62. The method of paragraph 60, wherein the scaffold is biodegradable.
63. The method of paragraph 60, wherein the scaffold comprises natural fibers, synthetic fibers, decellularized lung tissue, or combinations thereof.
64. The method of paragraph 60, wherein the scaffold comprises a substance that promotes differentiation of isolated human lung progenitor cells.
65. The method of paragraph 60, wherein the composition is formulated for aerosol delivery.
66. (a) culturing a human disease-specific airway cell population produced by in vitro differentiation of human disease-specific lung progenitor cells in the presence and absence of a candidate substance for treating a lung disease or disorder Stage,
(B) comparing the expression or activity of at least one marker up-regulated in the disease in the presence and absence of the candidate substance, or the expression or activity of at least one marker down-regulated in the disease Lungs in a subject if the expression or activity of at least one up-regulated disease marker decreases or the expression or activity of at least one down-regulated disease marker increases A method for screening a substance for treating a pulmonary disease or disorder comprising the step of identifying a candidate substance as a candidate for treating a disease or pulmonary disorder.
67. The method of paragraph 66, further comprising the step of differentiating the isolated human disease-specific lung progenitor cell population into a culture of human disease-specific airway cells prior to step (a).
68. Prior to step (a), further comprising the step of differentiating an induced pluripotent stem cell population derived from a subject having a lung disease or disorder into an isolated human disease-specific lung progenitor cell population 66. The method according to 66.
69. The method of paragraph 66, wherein the candidate substance comprises a small molecule, protein, polypeptide, antibody or antigen-binding fragment thereof, or a nucleic acid.
70. Human lung progenitor cells are human Nkx2.1 positive Sox2 positive proximal airway multipotent progenitor cells, human Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells, and human Nkx2.1 positive p63 positive multipotent 68. The method of paragraph 66, selected from the group consisting of sex airway basal stem cells.
71. Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells, BMP7, FGF7, WNT antagonist, and MAPKK / ERK antagonist, Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells, Nkx2.1 positive Sox9 positive Human Nkx2.1-positive Sox2-positive by a method comprising contacting at a sufficient time and concentration to differentiate into peripheral airway multipotent progenitor cells or Nkx2.1-positive Sox9-positive distal multipotent lung progenitor cells 71. The method of paragraph 70, wherein a lateral airway multipotent progenitor cell and a human Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cell are generated.
72. The method of paragraph 71, wherein the contacting step occurs for at least 4 days.
73. Nkx2.1 positive Tuj1 negative Pax8 negative cells, B27, BMP7, FGF7, and WNT antagonist, Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells, Nkx2.1 positive p63 positive multipotent airway basal stem cells 71. The method of paragraph 70, wherein human Nkx2.1 positive p63 positive multipotent airway basal stem cells are produced by a method comprising contacting at a sufficient time and concentration to differentiate into.
74. The method of paragraph 73, further comprising contacting Noggin with Nkx2.1 positive Tuj1 negative Pax8 negative cells.
75. The method of paragraph 73, wherein the contacting step occurs for at least 10 days.
76. (a) culturing Nkx2.1 positive Tuj1 negative Pax8 negative human lung progenitor cells in the presence and absence of candidate differentiation substances;
(B) comparing the expression or activity of at least one marker that is up-regulated upon differentiation of lung progenitor cells to a more differentiated state in the presence and absence of candidate substances, or lung progenitor cells Comparing the expression or activity of at least one marker that is down-regulated upon differentiation to a more differentiated state, wherein the expression or activity of at least one up-regulated differentiation marker decreases Or an increase in the expression or activity of at least one down-regulated differentiation marker indicates that the candidate substance can be used to induce differentiation of isolated human lung progenitor cells in the subject. A method for screening a substance that induces differentiation of human lung progenitor cells.
77. The method of paragraph 76, further comprising the step of differentiating embryonic stem cells or induced pluripotent stem cells into human lung progenitor cells prior to step (a).
78. The method of paragraph 76, wherein the candidate substance comprises a small molecule, protein, polypeptide, antibody, or nucleic acid.
79. Human lung progenitor cells are human Nkx2.1 positive Sox2 positive proximal airway multipotent progenitors, human Nkx2.1 positive Sox9 positive distal multipotent progenitor cells, and human Nkx2.1 positive p63 positive multipotency 77. The method of paragraph 76, selected from the group consisting of sexual airway basal stem cells.
80. Nkx2.1 positive Tuj1 negative Pax8 negative human lung progenitor cells, BMP7, FGF7, WNT antagonist, and MAPKK / ERK antagonist, Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells each Nkx2.1 positive Sox9 positive Human Nkx2.1 positive Sox2 positive by a method comprising contacting at a sufficient time and concentration to differentiate into proximal airway multipotent progenitor cells or Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells 80. The method of paragraph 79, wherein proximal airway multipotent progenitor cells and human Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells are generated.
81. The method of paragraph 80, wherein the contacting step occurs for at least 4 days.
82. Nkx2.1 positive Tuj1 negative Pax8 negative cells, B27, BMP7, FGF7, and WNT antagonist, Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells, Nkx2.1 positive p63 positive multipotent airway basal stem cells 80. The method of paragraph 79, wherein human Nkx2.1 positive p63 positive multipotent airway basal stem cells are produced by a method comprising contacting at a sufficient time and concentration to differentiate into.
83. The method of paragraph 82, further comprising contacting Noggin with Nkx2.1 positive Tuj1 negative Pax8 negative cells.
84. The method of paragraph 82, wherein the contacting step occurs for at least 10 days.
85. A kit for treating a pulmonary disease or disorder comprising the cell of paragraph 1, 5, or 9, a pharmaceutically acceptable carrier, and instructions for treating the pulmonary disease or disorder.
86. The kit according to paragraph 85, further comprising a scaffold.
87. A kit for screening a candidate substance comprising the cell according to paragraph 1, 5, or 8, one or more substances for detecting a lung-specific cell surface marker, and instructions thereof.
88. The kit of paragraph 87, further comprising a cell culture medium, growth factors, and / or differentiation agents.
89. (i) two or more of BMP4, FGF2, WNT, BMP7, FGF7, WNT antagonist, PI3 kinase inhibitor, activin A, Noggin, B27, and retinoic acid, optionally provided in unit dose;
(Ii) optionally a cell culture medium;
(Iii) one or more substances for detecting lung cell specific surface markers; and (iii) a kit for differentiating human stem cells into human lung progenitor cells, comprising instructions thereof.
90. A cell or tissue medium comprising B27, activin A, and ZSTK474.
91. The medium of paragraph 90, wherein the concentration of B27 is 1% to 5%.
92. The medium of paragraph 91, wherein the concentration of B27 is 2%.
93. The medium of paragraph 90, wherein the concentration of activin A is 10-40 ng / mL.
94. The medium of paragraph 93, wherein the concentration of activin A is 20 ng / mL.
95. The medium of paragraph 90, wherein the concentration of ZSTK474 is 0.2-0.5 μM.
96. The medium of paragraph 90 comprising a component of RPMI medium.
97. A cell or tissue medium comprising CHIR9902, PIK-75, dorsomorphin, and FGF2.
99. The medium of paragraph 97, wherein the concentration of CHIR9902 is in the range of 0.1-1 μM.
100. The medium of paragraph 97, wherein the concentration of PIK-75 comprises 0.01-0.1 μM.
101. The medium of paragraph 97, wherein the concentration of dorsomorphin comprises 1-5 μM.
102. The medium of paragraph 97, wherein the concentration of FGF2 comprises 10-100 ng / mL.
103. further comprising a drug selected from the group consisting of GF-109203X, Ro31-8220, Pp242, PIK-75, ZSTK474, PMA, carvedilol, corticosterone, triclabendazole, benproperin phosphate, phenothiazine, and methotrexate, 98. The medium of paragraph 97.
104.In human foregut endoderm cells, Wnt agonist, PIK3 kinase inhibitor, BMP antagonist, and GF-109203X, Ro31-8220, Pp242, PIK-75, ZSTK474, PMA, carvedilol, corticosterone, triclabendazole, A drug selected from the group consisting of benproperin phosphate, phenothiazine, and methotrexate for a period and at a concentration sufficient to differentiate human foregut endoderm cells into Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells A method of producing a human lung progenitor cell or human lung progenitor cell population that is Nkx2.1 positive, Tuj1 negative, and Pax8 negative, comprising a step of contacting.
105. The method of paragraph 104, wherein the contacting step occurs for at least 2 days.
106. The method of paragraph 104, wherein the Wnt agonist comprises CHIR9902.
107. The method of paragraph 106, wherein the concentration of CHIR9902 is in the range of 0.1-1 μM.
108. The method of paragraph 104, wherein the PI3 kinase inhibitor comprises PIK-75.
109. The method of paragraph 108, wherein the concentration of PIK-75 comprises 0.01-0.1 μM.
110. The method of paragraph 104, wherein the BMP antagonist comprises dorsomorphin.
111. The method of paragraph 110, wherein the concentration of dorsomorphin comprises 1-5 μM.
112. The method of paragraph 104, wherein the growth factor comprises FGF2.
113. The method of paragraph 112, wherein the concentration of FGF2 comprises 10-100 ng / mL.
114. A definitive endoderm cell or embryo comprising contacting iPSC or ESC with B27, activin A, and ZSTK474 for a period and at a concentration sufficient to each differentiate iPSC or ESC into definitive endoderm cells. A method of producing an endoderm cell population.
115. The method of paragraph 114, wherein production of definitive endoderm cells is determined by FOXA2 / SOX17 co-staining or by FACS analysis with a combination of cKit / CXCR4 and / or cKit / EpCAM.
116. The method of paragraph 114, wherein the concentration of B27 is between 1% and 5%.
117. The method of paragraph 116, wherein the concentration of B27 is 2%.
118. The method of paragraph 114, wherein the concentration of activin A is 10-40 ng / mL.
119. The method of paragraph 118, wherein the concentration of activin A is 20 ng / mL.
120. The method of paragraph 114, wherein the concentration of ZSTK474 is 0.2-0.5 μM.

  The discovery of embryonic stem cells (ES) cells and induced pluripotent stem cells (iPSC) provides an unprecedented opportunity to produce tissue-specific cell types that can be used in human disease modeling, drug screening, and patient-specific therapy Is given. Many iPSCs are currently produced by patients with lung disease, but the main obstacles to the practical development of human lung disease models using these cells are to convert them into lung progenitor cells Cannot be converted to differentiated lung epithelial cell types for subsequent therapeutic treatment, or cannot be characterized in the laboratory. Several attempts have been made to produce lung epithelial cells from mouse and human ES cells. In many cases, researchers have focused on the generation of type II alveolar cells (Van Vranken et al., 2005; Samadikuchaksaraei et al., 2006; Wang et al., 2007). Airway epithelial cells from pluripotent stem cells, despite the fact that airway diseases such as asthma, cystic fibrosis, bronchitis, and bronchogenic cancer are generally more common than alveolar diseases such as emphysema Few studies have targeted the differentiation of In addition, during lung primordium formation, the first two recognizable progenitor cells are multipotent embryonic lung progenitor cells and airway progenitor cells. Therefore, production of these two progenitor cells would be expected to allow differentiation of lung epithelial cells into each type.

  The majority of mouse airway epithelium and human airway epithelium are derived from four major cell types: basal stem cells adjacent to the basement membrane, secreted detoxifying Clara cells with P450 mechanism in the mouse, and mucus from the respiratory tree It consists of ciliated cells that drain and goblet cells that secrete mucus in response to injury or inflammation. Previous attempts to create functional lung epithelium from ES cells (Coraux et al., 2005; Van Haute et al., 2009) have shown that a limited number of cells are produced stochastically and after transplantation A mixed cell population containing undifferentiated pluripotent stem cells with a significant risk of teratoma formation was generated. Other attempts have utilized incompletely defined media to induce airway cell differentiation (such as exposure of human ES cells to tumor cell extracts; Roszell et al., 2009) or drugs Genetically modified pluripotent stem cells selected based on the presence of resistance genes are used (Wang et al., 2007). Unfortunately, genetic modification increases the potential for deleterious gene mutations in the resulting cells.

  In view of the difficulty of obtaining adult human airway stem cells to model human disease, the studies described herein generate normal and disease-specific lung epithelial cells from human normal and lung disease-specific iPSCs Pursuing to do. This is particularly important because mouse lung disease models often do not mimic the phenotype of human lung disease. A major example is Cftr knockout mice (Snouwaert et al., 1992; Clarke et al., 1992; Guilbault et al., 2006) that do not exhibit the cystic fibrosis-related lung pathology observed in human patients. In addition, since iPSC-induced epithelial cells reflect the pre-pathological state of lung cells, numerous secondary changes, such as changes associated with inflammation or infection, mask the effects of the first genetic abnormality in disease pathogenesis. There is no.

  The overall strategy described herein uses a stepwise differentiation approach. The mechanisms regulating mouse definitive endoderm regionalization, lung specification, and subsequent progenitor cell patterning and growth have been well studied (commented in Morrisey and Hogan, 2010). Initiation of lung specification within the endoderm is accompanied by Nkx2.1 expression and Sox2 downregulation along the dorsoventral axis of the intestine (Lazzaro et al., 1991; Minoo et al., 1999; Que et al. , 2009). Nkx2.1 is the earliest marker that distinguishes lung endoderm from the rest of foregut endoderm. Second, Sox2 expression increases again in future tracheal, bronchial, and bronchiolar regions, but markers of multipotent embryonic lung progenitor cell populations, Sox9, FoxP2, and ID2, are located at the distal lung bud tip Remains (Perl et al., 2005; Shu et al., 2007; Rawlins et al., 2009). Next, Nkx2.1 + Sox2 + airway progenitor cells present in the embryonic trachea differentiate into p63 + airway basal stem cells (Que et al., 2009). The mouse airway epithelium is very similar to most of the human airway epithelium (Rock et al., 2010; Rock et al., 2009; Evans et al., 2001) and therefore studies human airway disease Can be used as a model.

  We focused on the production of Nkx2.1 + thyroid endoderm and Nkx2.1 + lung endoderm lacking neural progenitor cells. A recent report using human ES cells demonstrated that differentiation strategies based on mimicking mouse intestinal organogenesis produced anterior foregut cells and Nkx2.1 + cells with an efficiency of up to 30% (Green et al., 2011). However, it has not been proved that these Nkx2.1 + cells are composed purely of lung progenitor cells. TUJ1 (nerve tissue marker) and PAX8 (thyroid tissue marker) expression was not assessed at the single cell level by co-antibody staining with Nkx2.1 antibody. Therefore, it is necessary to develop a new strategy to produce Nkx2.1 + progenitor cells that reflect only lung differentiation but not thyroid or brain differentiation, and the technology is widely applicable to human lung disease research In order to become, the technology must be applicable to human disease-specific iPSCs.

  As used herein, starting with mouse ES cells and then with normal and lung disease specific human iPSCs, definitive endoderm (DE), foregut endoderm, early lung endoderm, multipotent Nkx2.1 + embryonic lung An efficient and consistent stepwise differentiation method (FIG. 1) for producing progenitor cells and airway progenitor cells is described. We show that high doses of activin, transient WNT activation, and BMP4 inhibition over a period of time convert ES cells into definitive endoderm with high efficiency. Further inhibition of TGFβ alone is sufficient to move endoderm forward to Sox2 + foregut endoderm. These studies show that anterior migration of endoderm to foregut endoderm enhances terminal differentiation of Nkx2.1 + cells at a later stage of the strategy. BMP4, FGF2, and WNT are each required for induction of Nkx2.1 + immature embryonic progenitor cells. These early progenitor cells can mature in vitro into Nkx2.1 + Sox2 + proximal progenitor cells and Nkx2.1 + p63 + airway basal stem cells and can differentiate into mature airway epithelia.

Example 1A: A very efficient and universally applicable protocol for improving the efficiency of definitive endoderm Endoderm is one of the three primordial germ layers in the embryo. The endoderm forms the embryonic intestinal tract, which in turn produces the lung epithelium and gastrointestinal tract. Therefore, the first essential step in directing human iPSCs to the lung lineage is to produce endoderm cells, preferably with high efficiency. For various reasons, some cell lines do not produce definitive endoderm with high efficiency. For example, the currently published protocol (Mou et al., 2012; RPMI + 2% B27 + 100 ng / ml TGF-β agonist activin A + 5 μM LY-294002 PI3K inhibitor) is effective in several human iPSC and ESC strains, The efficiency of definitive endoderm is higher than 90%. In some “difficult” or “resistant” cell lines, this same protocol produces definitive endoderm that varies in efficiency from low (<30%) to moderate (40-70%). We have included LY-294002, ZSTK474 (Zenyaku Kogyo Co. ™), wortmannin, PI828 (PIramed Ltd. ™, Roche ™), NVP-BKM120 (Novartis ™), and PIK A number of PI3K inhibitors were screened, including -75 (Drug Discovery Research, Astellas Pharma Inc. ™), and ZSTK474 was determined to be the most potent for definitive endoderm production. In addition, the use of ZSTK474 can greatly reduce the dose of growth factor activin A (eg, from 100 ng / ml to 20 ng / ml, and in some cell lines to 10 ng / ml activin A) ). This protocol is therefore much more cost effective than previous protocols. Previously noted PI3 kinase inhibitors also include Promega (TM), Invivogen (TM), Sigma-Aldrich (TM), Cayman Chemicals (TM), Tocris (TM), and Cell Signaling Technologies (TM) It can be obtained from a vendor not limited to.

  This protocol provides a surprising 90% or more, including 91%, 92%, 93%, 94%, 95%, 96%, 97%, and even more, as determined by nuclear staining for transcription factors Foxa2 and Sox17 98% or more of human iPSC / ESC cells can be converted into definitive endoderm cells. Importantly, this protocol is very consistent from experiment to experiment (ie reproducible). In addition, this protocol can be universally applied to a large number of cell lines, including several “difficult” cell lines that have been determined to have low definitive endoderm production efficiency.

Protocol Summary : Human ESCs and iPSCs were grown in complete mTeSR1 medium in Geltrex ™ coated plates (low growth factor (RGF) basement membrane extract (BME) purified from mouse Engelbreth-Holm-Swam tumors) It was. Nourish the cells daily and differentiate at 65-80% confluence. For definitive endoderm differentiation, mTeSR1 was aspirated and cells were rinsed twice with warm RPMI 1640 medium to remove residual growth factors in mTeSR1. The medium was replaced with definitive endoderm differentiation medium (RPMI + 2% B27 + 20 ng / ml activin A + 0.2-0.5 μM ZSTK474). The medium was changed daily for a total differentiation period of 4 days. The efficiency of definitive endoderm was examined by FACS analysis with FOXA2 / SOX17 co-staining or a combination of cKit / CXCR4 and cKit / EpCAM.

  A diagram illustrating a protocol for producing definitive endoderm with high efficiency is shown in FIG. 13 herein.

Example 1B: By stage-specific TGFβ inhibition, naïve endoderm is localized to the anterior foregut endoderm and promotes differentiation of Nkx2.1 + cells Recent reports indicate that mouse ES cells are highly efficient in endoderm To move towards fate, a strategy for monolayer culture using synergistic activation of Nodal and Wnt-β catenin signaling and BMP4 inhibition over a period of time was described (Sherwood et al., 2011). This technique was adapted here to produce definitive endoderm (DE) from mouse ES cells. FIG. 7 shows an illustration of the strategy and approximate time for creating definitive endoderm. This protocol converted astonishing 80% -90% of cells to DE based on dual expression of endoderm transcription factors Foxa2 and Sox17 (data not shown). Next, it was examined whether or not newly prepared DE cells have the competence to generate Nkx2.1 + cells. After 2 days of exposure to serum-free medium containing BMP4, FGF2, and GSK3iXV (WNT agonist) (Figure 2A), less than 1% of the cells were observed to be Nkx2.1 + (data not shown) . However, more than 60% of the cells were Cdx2 +, indicating that the majority of cells were specialized for hindgut fate (data not shown). Since endoderm cells at this stage did not differentiate efficiently into Nkx2.1 + cells, we investigated whether the “forward movement” stage promotes lung fate specification. Snoeck and co-workers have shown that Noggin (BMP inhibitor) works synergistically with SB431542 (TGFβ inhibitor) to suppress posterior endoderm (Cdx2 +) fate in favor of anterior endoderm (Sox2 +) fate (Green et al., 2011). Since we have already incorporated BMP inhibition when making DE (Figure 7), whether continuous BMP inhibition is required for forward migration, or TGFβ inhibition alone for forward pattern formation. The question of whether it was enough was investigated. Endoderm was treated with a combination of activin (TGFβ agonist), A-83-01 (TGFβ antagonist), BMP4, and dorsomorphin (BMP4 antagonist) for 2 days (FIG. 2B). The number of FoxA2 + Sox2 + anterior endoderm cells was compared to the total number of FoxA2 + endoderm cells (FIG. 2B). Treated cells were further exposed to a BMP4 / FGF2 / WNT agonist cocktail for an additional 2 days to examine their competence to generate Nkx2.1 + cells (FIG. 2B). The results show that TGFβ inhibition alone was sufficient to increase FoxA2 + Sox2 + anterior endoderm (40% -55% compared to <0.1% with medium alone, FIG. 2B), And TGFβ inhibition enhances the competence of endoderm cells forming Nkx2.1 + cells (approximately 7% to 13% compared to <1% without localization, FIG. 2B). It was observed that sustained activin treatment yielded few FoxA2 + Sox2 + cells and fewer Nkx2.1 + cells (Figure 2B), indicating that exposure to activin for optimal duration is limited. It plays a role in the efficiency of crystallization. Similarly, generation of FoxA2 + Sox2 + cells did not require further BMP4 inhibition, and it was also found that Nkx2.1 + cells ultimately obtained by further BMP4 inhibition could be slightly lower in proportion (FIG. 2B). Similarly, when BMP4 is present for 5-7 days, it is also observed that fewer neuroectodermal markers Tuj1 are present (data not shown), which indicates that BMP4 suppresses nerve restraint, Consistent with findings that promote non-neural lineage differentiation from ESCs (Zhang et al., 2010). FGF and WNT signaling has been shown to maintain hindgut identity and actively suppress foregut endoderm differentiation (Wells and Melton, 2000; Ameri et al., 2010). We tested whether their inhibition enhanced forward migration with TGFβ inhibitors. Subsequent experiments revealed that the FGF antagonist (PD173074) induced cell death, whereas the WNT antagonist (IWR-1) did not increase the FoxA2 + Sox2 + population (data not shown).

Example 2: Murine ESC-derived Nkx2.1 + cells are proliferative, lacking neural and thyroid markers, and include distal multipotent lung progenitor cells and proximal airway progenitor cells
Nkx2.1 is not a specific marker of the lung and its expression is also found in the thyroid and ventral forebrain. It was therefore important to determine the identity of Nkx2.1 + cells produced in the culture system used herein. Unfortunately, there are no reliable markers of the lung lineage at embryonic stage E9 that are not expressed in the brain or thyroid. Sp-C (surfactant protein C) is the most specific marker of lung epithelial progenitor cells but is not detected until E10-E11 (Wert et al., 1993). SOX9 and FOXP2 are actually co-expressed with NKX2.1 in distal lung multipotent epithelial progenitor cells (data not shown) and are not found in the brain or newly specialized thyroid (data shown) Not), but its expression is not evident until after E11-E12. On the other hand, SOX2 is expressed in E9 lung endoderm (data not shown) and later expressed in airway epithelial progenitor cells (data not shown), but SOX2 is also NKX2 in the ventral forebrain. It can also be found in 1-expressing cells (Figure 8 and data not shown). Thus, at the earliest stages of lung endoderm specification (E8.75-E9), all of these markers (SPC, SOX2, SOX9, and FOXP2) reliably identify lung cells uniquely Can not be used for. In contrast, TUJ1 expression in Nkx2.1 + cells in the ventral forebrain began at E8.0 and continued to exist in the ventral forebrain and not in the thyroid or lung (Figure 8 and data). Tuj1 expression is a specific indicator of neuronal identity in the NKX2.1 domain. PAX8 expression is detected in the primordial thyroid at E8.75 at the time of thyroid specification (data not shown) but not in the lung or brain. Therefore, PAX8 can be used as a specific indicator of thyroid cell identity in the NKX2.1 domain (FIG. 8 and data not shown). In summary, we conclude that at the initial specification stage, Nkx2.1 + / Tuj1- / Pax8- cells may be considered to have a lung cell lineage. Therefore, to exclude nerve and thyroid identity, ESC-induced Nkx2.1 + cells were examined for TUJ1 and PAX8 expression (data not shown). All ex vivo differentiated Nkx2.1 + cells were co-stained with Foxa2, as expected for any Nkx2.1 + cells in the embryo (thyroid, lung, and ventral forebrain) (data not shown) ). A small subset of Tuj1 + cells was detected in culture, but these neurons did not overlap with Nkx2.1 + cells (data not shown). We did not detect any Pax8 + cells in the culture (data not shown). Therefore, these data indicate that Nkx2.1 + cells differentiated in vitro represent lung endoderm cells. These Nkx2.1 + cells are proliferative and more than half of them express Ki67 (data not shown).

  Data from immunofluorescence experiments indicate that ESC-induced Nkx2.1 + cells lack proliferative and thyroid markers and are proliferative and possess proximal and distal lung endoderm markers. When immunofluorescent staining for FOXA2, TUJ1, and PAX8 was performed, Nkx2.1 + cells were positive for endoderm marker FOXA2, negative for neuroectodermal marker TUJ1, and negative for thyroid marker PAX8 Was shown (data not shown). Nkx2.1 + cells were proliferative as evidenced by co-staining with Nkx2.1 and KI67 (data not shown). Immunofluorescence staining also showed the presence of a subpopulation of Nkx2.1 + cells that were positive for SOX2 (airway progenitor cell marker) and FOXP2 / SOX9 (multipotent lung progenitor cell marker) (data not shown) .

  In mice, Sox2 is rapidly down-regulated in this same foregut pre-lung endoderm during N9x2.1 expression in the pre-lung endoderm during the lung specification process at E9 (data shown Not) Thus, the earliest lung endoderm cells are Nkx2.1 + and Sox2-low cells. Shortly thereafter, during the branching morphogenesis process, persistently high levels of SOX2 expression are detected in the proximal airway epithelial progenitor cells of future trachea, bronchi and bronchioles (data not shown). Therefore, airway progenitor cells are Nkx2.1 + Sox2 +.

  In contrast, SOX9 and FOXP2 are expressed only in distal terminal multipotent lung progenitor cells (data not shown), which allows SOX9 and FOXP2 markers to be proximal airway progenitors as described above. Its absence in cells or thyroid or brain provides a marker for uniquely identifying a distinct population of multipotent embryonic lung progenitor cells within the NKX2.1 domain. Therefore, we observed that ES cell-derived Nkx2.1 + cells on day 9 were proximal airways (Nkx2.1 + Sox2 +) and distal multipotency (Nkx2.1 + Sox9 + or Nkx2. 1 + FoxP2 +) was checked whether it contains both progenitor cells. Nkx2.1 + cells generated from 10 independent experiments were counted. The average ratio of airway progenitor cells (Nkx2.1 + Sox2 +) is 1.8% ± 0.8% in total Nkx2.1 + cells, the ratio of distal multipotent progenitor cells Nkx2.1 + Sox9 + or Nkx2.1 + FoxP2 + Were 4.8% ± 2.5% and 6.6% ± 3.1% in total Nkx2.1 + cells, respectively. The remaining Nkx2.1 + cells (neither thyroid nor nerve) are those with minimal SOX2 expression and may reflect the early prelung endoderm. Thus, the majority of Nkx2.1 + cells at day 9 probably represent early lung endoderm that has not been specified to airway progenitor cells or to distal terminal multipotent lung progenitor cell populations. Immunofluorescence indicates that the cells are enriched for Nkx2.1 + Sox2 +, Nkx2.1 + Sox9 +, and Nkx2.1 + FoxP2 + cells (data not shown). Overall, these results show that at day 9 ESC-induced Nkx2.1 + cells are immature and most lack proximal and distal progenitor cell markers and represent early lung endoderm cells. It is implied. Although rare, the presence of Nkx2.1 + Sox2 +, Nkx2.1 + Sox9 +, and Nkx2.1 + FoxP2 + cells can cause ES cell-induced Nkx2.1 + lung endoderm cells to later differentiate into mature epithelial cells It was shown that it was differentiating into progenitor cells of the respiratory tract and multipotent lung progenitor cells.

  Expression of lung and other anterior cell fate lineage genes including FoxN1 (thymus), Pax8 (thyroid), and Pax9 and Tbx1 (pharyngeal sac endoderm) was analyzed by real-time qPCR (FIG. S4A). As expected, the results showed that Sox2 is down-regulated when pluripotent cells differentiate into definitive endoderm (DE). Later, Sox2 levels increased according to the expectation that the anterior endoderm expresses Sox2. Nkx2.1, Sox9, and FoxP2 expression was minimal in ESC, definitive endoderm, and anterior endoderm, but greatly increased (10-20 fold) after stimulation with FGF2 / WNT / BMP4 induced cocktail Consistent with previous results demonstrating a combination of these lung-specific markers by antibody staining (data not shown). Similarly, FoxN1, Tbx1, Pax8, and Pax9 expression was low in ESC, definitive endoderm, and forward migrating endoderm. However, a modest increase in Pax9 and Tbx1 expression was observed in growth factor-induced anterior foregut cells, but FoxN1 and Pax8 expression was still very low. Despite increased Pax9 and Tbx1 gene expression, we did not detect any reproducible staining for these proteins by immunofluorescence (data not shown). All these results are consistent with the finding that the combination of FGF, BMP4 and WNT signaling primarily drives the differentiation of lung-specific Nkx2.1 + progenitor cells from the anterior endoderm.

Example 3A: BMP4, FGF, and WNT signaling are each required for NKX2.1 induction in vitro and repeat mouse lung development
We tested whether BMP4, FGF2, and WNT signaling were each required to specify lung endoderm from the anterior foregut in the in vitro mouse ESC differentiation system. To do this, forward migrating endoderm cells were exposed to a combination of BMP4, FGF2, and WNT agonist and antagonist (FIGS. 3A and B). It was found that BMP4 alone can induce NKX2.1 expression. In contrast, FGF2 and / or GSK3i were not sufficient for NKX2.1 induction without BMP4, indicating that BMP4 signaling is required for lung specification (FIGS. 3B and C). Interestingly, in the presence of FGF and WNT antagonist, BMP4 could no longer induce Nkx2.1 + cells. This result implies endogenous secretion of FGF and WNT ligands. Similarly, FGF and WNT activation suggests that it is required for BMP4-dependent Nkx2.1 specification (FIGS. 3B and C). Despite putative endogenous FGF signaling, increasing FGF2 concentration up-regulated Nkx2.1 + cells (FIGS. 3B and C). Compared to FGF2 signaling, excessive WNT activation was detrimental, Nkx2.1 + cells decreased (FIGS. 4B and C), and hindgut Cdx2 + cells increased (data not shown).

  BMP2, 4, and 7 are the most studied BMP family members. Both BMP2 and BMP4 transduce signals through the type I receptor (ALK3), while BMP7 binds to a different type I receptor (ALK2) (commented by von Bubnoff and Cho, 2001; Chen et al., 2004 Sieber et al., 2009; Miyazono et al., 2010). The effects of BMP2 and BMP7 were compared to BMP4 with respect to the induction of Nkx2.1 + cells from the anterior endoderm. The results showed that BMP2 was less efficient than BMP4 in inducing Nkx2.1 + cells, but BMP7 (10 ng / ml) had no effect (data not shown). Even with increasing concentrations (> 100 ng / ml), BMP7 still failed to generate Nkx2.1 + cells (data not shown). This indicates that BMP7 signaling via the ALK2 receptor was not necessary for NKX2.1 induction. In standard BMP2 / 4 signaling, BMP2 / 4 binds to the BMP receptor I / II complex and phosphorylation of Smad1 / 5/8 occurs, forming a heterocomplex with Smad4. These complexes translocate to the nucleus and activate target gene expression (von Bubnoff and Cho, 2001; Chen et al., 2004; Sieber et al., 2009; Miyazono et al., 2010). In addition to Smad1 / 5 / 8-mediated transcription, BMP-induced receptor complexes can activate the mitogen-activated protein kinase (MAPK) pathway by ERK, JNK, or p38 (Kozawa et al., 2002) . With dorsomorphin (pSmad1 / 5/8 inhibitor) and PD98059 (MAPKK / ERK inhibitor), dorsomorphin completely abolishes the production of Nkx2.1 + cells, but PD98059 is only partially Nkx2. It was observed that only the 1+ cell ratio was reduced (data not shown). Significant cell death was observed in the presence of PD98059 and therefore a decrease in Nkx2.1 + cells could not rule out the possibility of apoptosis. In addition, Smad and MAPK-mediated pathways can be integrated (Aubin et al., 2004) and the decrease of Nkx2.1 + cells in the presence of PD98059 may also be due to down-regulation of Smad-dependent signaling There is. Overall, it was concluded that a Smad-dependent BMP2 / 4 signaling cascade is required for NKX2.1 specification from foregut cells (Figure 4).

Example 3B: Nkx2.1 + differentiation to generate lung progenitor cells
High-throughput chemical screening was used to identify compounds capable of promoting Nkx2.1 + lung progenitor cells and to identify novel signaling pathways that control human lung specification.

  In order to increase the production efficiency of Nkx2.1 + lung progenitor cells, we identified low molecular weight compounds and FDA approved clinical drugs that can promote NKX2.1 positive lung progenitor cell differentiation. An unbiased high-throughput chemical screen was performed on the kinase pathway inhibitor library and the NIH clinical drug library. The chemical screening platform is shown in FIG. 14A. Screening was performed at the stage of conversion of Foxa2 + Sox2 + anterior endoderm to Nkx2.1 + lung endoderm.

  From the kinase compound set (240 compounds), at least 5 molecules were identified that produced a statistically significant (more than 3-fold) increase in the proportion of Nkx2.1 + cells, and the NIH Clinical Collection (400 compounds) ) Identified at least 6 drugs (FIG. 14B). These Nkx2.1 + cells are further stained in different experiments to exclude expression of the neural lineage marker Tuj1 and thyroid lineage marker Pax8. In addition, all positive candidates identified in the initial screen have been evaluated for drug persistence and dose effects to increase lung progenitor cell formation, including cytotoxicity induction, Ki67 staining cells It has been evaluated for other effects including proliferation. Since these compounds are annotated, we have found that there are a number of positive hits that target the same pathway, such as the PI3K and PKC-related signaling pathways. Without wishing to be bound by theory, these data indicate that these biological pathways are actively involved in human lung organogenesis. Similarly, it was also determined that BMP4 is not important for human lung progenitor cell differentiation from iPSC, indicating that lung development is species-specific in humans and mice. Inhibiting BMP4 creates lung progenitor cells with high NKX2.1 expression levels, but adding BMP4 decreases NKX2.1 expression without affecting the total number of NKX2.1 + lung progenitor cells Was decided.

  In addition to positive hits, some compounds have been found to reduce or even block NKX2.1 + cell production. Some of these negative compounds are MEK1 / 2 antagonists. Without wishing to be bound by theory, these data indicate that the MEK1 / 2-related signaling pathway plays an important role in human lung organogenesis and lung progenitor cell differentiation from iPSCs. For example, endoderm-specific deletion of MEK1 / 2 results in pulmonary atrophy in mice (unpublished data). In the in vitro differentiation system, we show that activation of MEK1 / 2 (such as by phorbol-12-myristate 13 acetate or PMA and other MEK1 / 2 agonists) stimulates NKX2.1 + cell production. I found.

  After studying the additive and synergistic effects of positive hits, Nkx2.1 + lung progenitor cells from a number of iPS cell lines without xenogeneic components were maintained in order to maintain cells suitable for future clinical applications. In order to create highly enriched populations (up to 85% -90%), a strong and economically effective new protocol was devised. This exemplary differentiation medium consists of one Wnt agonist (eg, 0.5 μM CHIR9902), one PI3 kinase inhibitor (eg, 0.02-0.05 μM PIK-75), one drug used clinically (eg, 0.02 -0.05 μM methotrexate), one BMP antagonist (eg, 2-4 μM Dorsomorphin) and one growth factor (eg, 10-50 ng / ml FGF2). FIG. 12 is a summary of the newly developed stepwise airway progenitor differentiation protocol from iPSC to airway progenitor cells. One skilled in the art can make many other protocols from this basic protocol (RPMI + 2% B27 + 10-50 ng / ml FGF2 + 0.5 μM CHIR9902 + 2 + 4 μM Dorsomorphin) with the compounds described in FIG.

  Based on current media costs, we have determined that the cost of differentiation media from iPSCs to lung progenitor cells (stage I to stage III) is a fraction of the original cost reported by Mou et al., 2012. Although calculated to be 10%, this protocol increased the efficiency of NKX2.1 + cell production.

  In addition, the inventors have demonstrated that this approach is effective in a large number of human iPS cells and ESC cells. Stage IV matures immature lung progenitor cells into NKX2.1 + SOX2 + p63 + CK5 + airway stem cells, as well as transforms such populations into functional assays, disease modeling, and drug screening, for example. , Can be used to grow repeatedly and / or increase the amount of cells.

Example 4: Combination of BMP7 and FGF7 signaling and WNT and MAPKK / ERK inhibition creates proximal airway progenitor cells As demonstrated previously herein, most ESC-induced Nkx2 at day 9 .1+ cells were immature lung endoderm cells. Therefore, these cells were differentiated to produce more mature Nkx2.1 + Sox2 + embryonic airway progenitor cells. We selectively applied signaling factors to generate airway stalk progenitor cells from immature lung Nkx2.1 + cells (FIGS. 5A and B). Cells were switched to “proximal induction” medium prepared with RA-added B27, BMP7, FGF7, IWR-1 (WNT antagonist), and Noggin on day 9 (data not shown). After 2-3 days, an increase in Nkx2.1 + Sox2 + airway progenitor cells was observed. The ratio was approximately 10% of the total number of Nkx2.1 + cells compared to 1% to 2% before treatment. In different experiments where individual growth factors were removed, WNT antagonism was found to have the most significant effect on the production of Nkx2.1 + Sox2 + cells. Addition of dorsomorphin (BMP antagonist) instead of Noggin at this stage had little effect on the number of Nkx2.1 + Sox2 + cells. Interestingly, the small molecule PD98059 (MAPKK / ERK inhibitor) enhanced the production of Nkx2.1 + Sox2 + airway progenitor cells by 18% of the total number of Nkx2.1 + cells (data not shown). These data indicate that airway progenitor cells are formed after inhibition of MAPKK / ERK-related pathways. Most notably, we began to detect a small fraction of Nkx2.1 + p63 + cells (approximately 1% -4% of the total number of Nkx2.1 + cells) on days 11-12 ( Figure 6D). Increasing the culture period from 2 days to 5 days or longer in “proximal induction” medium resulted in higher percentages of Nkx2.1 + Sox2 + and Nkx2.1 + p63 + cells (data shown) Absent). The production of Nkx2.1 + Sox2 + and Nkx2.1 + p63 + cells still needs to be optimized, but these results show that ESC-induced Nkx2.1 + cells become proximal airway progenitor cells and induced airway basal cells And prove that it can be differentiated. Approaches to isolate specific subpopulations of these cells, such as Nkx2.1 + Sox2 + cells, or Nkx2.1 + Sox9 + cells and Nkx2.1 + p63 + cells, are reporter gene constructs such as Sox2, Sox9, or Transfecting cells with a GFP reporter construct driven by the p63 promoter. If GFP is expressed in these situations, it is identified that the cell is permissive for expression of the factor of the invention. It is contemplated that these tests can be performed on cells at limiting dilution. For example, anterior foregut endoderm cell populations are induced to differentiate into Nkx2.1 + Tuj1-Pax8-multipotent lung progenitor cells described herein and then airway progenitor cells as described Treatment to differentiate into can be performed. After an appropriate period of induction conditions, the population was subjected to limiting dilution and plated one cell per well in a microtiter dish. Isolated cells are grown clonally in culture, and a portion of the expanded cells are induced airway progenitor cell markers such as Nkx2.1, Sox2, Sox9, p63, or even more distal differentiation markers, For example, the expression or co-expression of CCSP, FoxJ1, etc. can be tested, for example by immunofluorescence. In this way, isolated populations of various progenitor cells can be prepared.

  One skilled in the art will recognize that alternative methods of producing an isolated cell population can also be used with the methods described herein. In some embodiments, chemicals or combinations of chemicals can be used to enhance cell differentiation efficiency in culture to increase the number of desired cell types. Such enhanced cell differentiation efficiency allows the production of an isolated population of the desired human lung progenitor cell type, for example by positive selection for the desired cell phenotype or by selective killing of the unwanted cell phenotype Become. Alternatively, cells can be expressed so that the cells express GFP upon differentiation to the desired human lung progenitor cell phenotype, so that cells of a given phenotype can be isolated using, for example, FACS sorting. It can be genetically modified. In addition, one of skill in the art can generate antibodies (eg, monoclonal antibodies) against specific cell surface markers or marker combinations. Such antibodies or combinations of antibodies can be used to purify the desired cell type from the cell population.

Example 5: ESC-induced Nkx2.1 + cell population can differentiate into mature airway epithelium when transplanted in vivo Nkx2.1 + lung progenitor cells derived from pluripotent stem cells are useful Must possess the ability to generate functional respiratory epithelium. Therefore, mouse ESC-derived Nkx2.1 + progenitor cells were tested for their ability to form mature respiratory epithelium by subcutaneous engraftment (data not shown). The assay evaluated the ability of Nkx2.1 + cells to differentiate within a mixed cell population. 20,000-50,000 cells were suspended in 50% Matrigel and injected subcutaneously into immune deficient mice. Twenty to thirty days after injection, the engrafted tissue was removed for examination. Differentiation of airway epithelium from mouse ESC-induced lung endoderm was observed during subcutaneous engraftment. Immunofluorescence staining shows that some Nkx2.1 + cells are positive for SOX2 (data not shown). Immunofluorescence staining further showed that ESC-induced Nkx2.1 + cells differentiated into p63 + airway basal stem cells, CC10 + Clara cells, FoxJ1 + ciliated cells, and Muc5ac + goblet cells (data not shown).

  Many epithelial spheres were formed in the graft, and some of these spheres were observed to contain Nkx2.1 expressing cells. Some Nkx2.1 + spheres include Sox2 + proximal airway epithelial cells (data not shown), p63 + basal stem cells, CC10 + Clara cells, FoxJ1 + ciliated cells, and Muc5ac + mucin secreting cells (data not shown) Markers of mature airway epithelial cells (data not shown) were detected.

  Triple immunofluorescence staining with confocal imaging demonstrated that the sphere contains more than one marker of mature airway epithelium. Such mature airway epithelial markers have never been detected in ESC-induced teratomas. Previously published basal stem cell differentiation using a three-dimensional sphere formation assay produced cilia and basal cells, but not Clara cells (Rock et al., 2009). Although we have distal pulmonary multipotent cells (Nkx2.1 + Sox9 + and Nkx2.1 + FoxP2 + cells) in the initial cell mixture, such as PRO-SPC, PRP-SPA, and AQUAPORIN5 No type I and type II alveolar cell markers were detected. Overall, it was concluded that ESC-induced Nkx2.1 + cell-containing populations can perform airway epithelial cell differentiation.

Example 6: Efficient and reproducible stepwise approach to generate NKX2.1 + lung cells from human cystic fibrosis iPS cells Next, using a similar stepwise differentiation approach, cystic fibrosis ( CF) We investigated whether lung airway progenitor cells could be prepared from disease-specific human iPSCs (Fig. 6). All differentiation stages were performed under conditions that did not contain xenogeneic components. In addition, two of the CF iPSC strains were created using modified RNA (RiPS) rather than viral code reprogramming factors (Warren et al., 2010), so the gene was not modified and this is possible It was another advantage for potential future clinical use. CF iPSCs were maintained on Geltrex coated plates in complete mTeSR1 medium. High yields of definitive endoderm (DE) were obtained after 3-4 days treatment in RPMI 1640 medium in the presence of activin (100 ng / ml) and the PI3 kinase inhibitor LY294002 (5 μM). More than 85% to 90% of cells coexpress the transcription factors SOX17 and FOXA2 on day 4, and DE is highly efficient from RiPS cell lines that are complex heterozygous for the CFTR mutant alleles Δ508 and G551D (Data not shown). This protocol also includes three cystic fibrosis iPSC lines that are homozygous for the △ 508 allele, the wild type human BJ RiPS cell line (Warren et al., 2010), and the HUES-3 and HUES-9 human ESC lines. Has been successfully applied to other human pluripotent stem cells including (data not shown).

  By using forward migration conditions similar to Green et al., Induction of SOX2 expression in DE cells was reduced from 50% to 60% after 4 days of treatment with A-83-01 (TGFβ antagonist; data not shown) Obtained with efficiency. Noggin (a BMP4 antagonist) was found to be actually not necessary for this forward movement. Using similar combinations of growth factors and agonists identified in studies on mouse ES cells (BMP4, FGF2, and GSK3iXV), NKX2.1 + cells can be generated with an efficiency of 10% to 30% (data Is not shown). These NKX2.1 + cells were negative for TUJ1 and PAX8 (data not shown), demonstrating that they are not nerve or thyroid identity cells. In addition, these subpopulations of NKX2.1 + cells were also positive for SOX2 and SOX9 (data not shown). Both of these markers suggest the presence of restricted airway progenitor cells (NKX2.1 + SOX2 + cells) and multipotent embryonic lung progenitor cells (NKX2.1 + SOX9 + cells).

  When the expression of various forward cell fate genes was quantified by qPCR (FIG. 9), a similar gene expression pattern was observed compared to the expression pattern observed during mouse ES cell differentiation. SOX2 was down-regulated after differentiation into definitive endoderm and then increased as expected after forward migration. A dramatic upregulation (20-30 fold) of NKX2.1, SOX9, and FOXP2 expression in forward migrating endoderm cells was observed after FGF2 / BMP4 / WNT induction. The expression of other anterior cell fate genes, including PAX8 (thyroid endoderm), PAX9 (pharyngeal endoderm), and TBX1 (pharyngeal endoderm ahead of the lung / esophagus), without any expression of FOXN1 (thymic endoderm) Up-regulated very modestly. Taken together, the endoderm differentiation spectra were remarkably similar on mouse and human platforms.

  Finally, human RiPS cell-derived NKX2.1 + mixed cell populations were engrafted subcutaneously (data not shown). Many spheres were formed in the engrafted tissue after 30 days subcutaneously in immune deficient recipient mice. In NKX2.1 + spheres, some of NKX2.1 + cells co-expressed p63, indicating that these NKX2.1 + cells matured into airway basal stem cells (data not shown) ).

Example 7: Summary To differentiate pluripotent stem cells into lung multipotent progenitor cells (Nkx2.1 + Sox9 + and Nkx2.1 + FoxP2 +) and airway progenitor cells (Nkx2.1 + Sox2 +) Report a step-by-step strategy. We show that subsequent differentiation of Nkx2.1 + lung cells was enhanced by mimicking localization of the embryonic foregut. This credits the idea that optimization of each individual stage during embryogenesis results in improved differentiation efficiency at each subsequent stage. Some of these Nkx2.1 + lung endoderm cells terminally differentiated into multipotent embryonic lung progenitor cells and airway progenitor cells, but this finding has not been previously reported. Furthermore, engraftment of restricted Nkx2.1 + lung progenitor cells produced a specific mature cell marker for airway epithelium. The inventors then adapted this strategy to produce disease-specific lung progenitor cells from human cystic fibrosis iPS cells and other human pluripotent stem cell lines, and thus human lung A new platform for analyzing disease was created.

  In addition, the ESC system can be used as a discovery tool to examine stages in lung development. Similarly, it has also been demonstrated that FGF signaling in addition to BMP and WNT signaling is required to induce NKX2.1 in current culture systems, which is near the foregut lung primordium. This finding has been considered difficult to define in the mouse gene system due to the presence of many FGFs. Analysis of the mechanism of action of BMP4 signaling also reveals new findings that BMP4 signaling occurs through a Smad-dependent pathway and that pharmacologically adjusting Smad enhances Nkx2.1 + lung cell differentiation became. The inventors have also shown that a carefully set period of antagonism or agonism of the same pathway is essential to drive optimal differentiation. For example, the initial addition of WNT signals, which are later required to induce Nkx2.1 + lung cells at the endoderm stage, enter the hindgut intestinal epithelium, which is refractory to Nkx2.1 + cell formation. The germ layers are localized (Spence et al., 2011; Cao et al., 2011; Sherwood et al., 2011). Thus, not only the combination of signaling cascades, but also their exact timing is essential to repeat in vivo differentiation. Thus, the system described herein is a useful auxiliary platform for defining mechanisms of lung cell differentiation not previously described using mouse developmental genetics.

  This study demonstrated for the first time that Nkx2.1 + Sox2 + proximal airway progenitor cells and Nkx2.1 + p63 + double positive airway stem cells are derived from both human and mouse pluripotent stem cells. Zaret and co-workers have shown that the anterior gut exhibits an epigenetic pattern that regulates the ability of gut endoderm to differentiate into specific tissues, despite the presence of the correct growth factor (Zaret et al., 2008). It is suggested to include. It would be important to examine whether the endoderm prepattern based on epigenetic factors can be adjusted to enhance lung progenitor cell specification.

  The present inventors succeeded in differentiating the mixed cell population into airway epithelium by engrafting the mixed cell population subcutaneously in immune-deficient mice. Indeed, other cells in this mixed population may be required to induce airway progenitor cells to form respiratory spheres.

  In summary, in this specification, from mouse ESCs, DE production, anterior migration of the endoderm to the foregut, and induction of the earliest lung Nkx2.1 + endoderm and multipotent Nkx2.1 + Sox9 + lung progenitor cells Strong, generally applicable protocols for inducing Nkx2.1 + Sox2 + embryonic airway progenitor cells and Nkx2.1 + p63 + airway stem cells are provided. This strategy was adapted to generate disease-specific lung progenitor cells from human cystic fibrosis iPSC, thus establishing a new platform for analyzing human lung disease. This allows for disease modeling, drug screening, gene rescue of mutant CFTR genes (using homologous recombination, zinc finger nuclease, or TAL effector nuclease) and autologous transplantation. Furthermore, this strategy can basically be applied to any genetically affected lung disorder in humans. Given the large amount of data that is emerging in the genetics of general human lung disease, this platform will also serve as a starting point for testing the biological significance of candidate genes in airway epithelia. Finally, the ability to generate a large number of lung epithelial cells may allow biochemical and proteomic experiments that were not possible before due to the limited supply of human lung epithelial cells.

Example 8: Experimental technique Mice and human cell lines: NOD-SCID IL2Rγ null (NOD / SCID / IL2Rγ-/-) immunodeficient mice were purchased from Jackson Laboratory. Animal techniques were performed in accordance with Massachusetts General Hospital (MGH) and national guidelines and regulations and were approved by the MGH Institutional Animal Care Committee (IACUC). The use of human cystic fibrosis-inducing pluripotent and embryonic cell lines (Hues-3 and Hues-9) has been reviewed and approved by the MGH Embryonic Stem Cell Research and Surveillance Committee (ESCRO) and IRB.

  Mouse endoderm forward migration and Nkx2.1 cell differentiation: Mouse definitive endoderm was made as described herein. To move the endoderm forward, cells were split and replated on plates pre-coated with 804G-conditioned medium on day 5 and thereafter, D0 medium + 0.5-1 μM A8301 (CalBiochem, 616454) For 2 days. The medium was then rinsed twice with D0 medium and switched to D0 medium supplemented with 50 ng / ml BMP4, 100 ng / ml FGF2 (GIBCO, PHG0026) and 5-10 nM GSK3iXV for an additional 2-3 days. To make Nkx2.1 + Sox2 + proximal progenitor cells, cells are rinsed twice with D0 medium, RA-added B27, 50 ng / ml BMP7, 50 ng / ml FGF7, 100 nM IWR-1, and 1-2 μM Switching to D0 medium containing PD98059 for 2 days or longer.

  Human iPSC culture and differentiation: Human iPSCs were maintained in complete mTeSR1 medium (Stemcell) on Geltrex coated plates. 3-4 in RPMI-1640 medium in the presence of 2% B27 supplement without vitamin A (GIBCO, 12587-010), activin A (Peprotech, 100 ng / ml), and the PI3 kinase inhibitor LY294002 (5 μM) High yields of definitive endoderm progenitor cells were obtained after daily treatment. To produce foregut endoderm cells, definitive endoderm is 2% B27, 500 nM A in the presence or absence of Noggin (BMP4 antagonist, 100 ng / ml) or Dorsomorphin (2-5 μM). Treated with RPMI-1640 medium containing -83-01 (TGFβ antagonist) for 4 days. Then, to induce Nkx2.1 from endoderm cells, the cells were placed in RPMI-1640 medium containing 2% B27, 50 ng / ml BMP4, 100 ng / ml FGF2, and 5 nM GSK3iXV for 4 days or more Long-term exposure.

  Immunofluorescence: At each differentiation stage, cells are fixed with fresh paraformaldehyde (4%) for 15 minutes at room temperature, rinsed with PBS, washed with PBS + 0.2% Triton X-100, diluted in PBS + 1% BSA The primary antibody was incubated at 4 ° C. overnight (> 16 hours). Following incubation, cells were rinsed 4 times with PBS + 0.2% Triton X-100 and incubated with secondary antibody for 2 hours at room temperature. Images were visualized using an Olympus IX71 inverted fluorescence microscope or a Nikon A1 confocal laser microscope. The primary antibodies used are summarized in the table below.

(Table 1) List of primary antibodies

  Secondary antibodies were purchased from Invitrogen (AlexaFluor-549 and AlexaFluor-488). Quantification was performed by counting at least 5 random fields at 20x magnification and calculating mean and standard deviation.

  Embryo collection, sectioning, and staining: Embryos or lungs at the desired embryogenesis stage were removed and fixed with fresh 4% PFA at 4 ° C. for 6 hours or overnight. Tissues were then rinsed with PBS for 3 minutes (5 minutes each time) and incubated overnight at 4 ° C. in 30% sucrose in PBS. The tissue was immersed in OCT for 1 hour and then frozen in OCT to prepare a frozen section having a thickness of 7 μm. Glass slides were stained with primary and secondary antibodies as described above.

Mouse ESC and human iPS-induced Nkx2.1 + lung progenitor cell differentiation after subcutaneous engraftment: 2-5 × 10 ⁵ cells in 200 μl of 1: 1 mixture of low growth factor Matrigel (BD Biosciences, 354230) Advanced DMEM And then injected subcutaneously into NOD-SCID IL2Rγ null mice. Tissues are harvested and examined after 20-30 days, fixed in cold 4% paraformaldehyde overnight, rinsed twice with PBS, soaked in 30% sucrose for 2-3 hours, and then embedded in OCT. 7 μm sections were prepared. Glass slides were examined for airway epithelial cells based on NKX2.1 expression. ESC-induced Nkx2.1 + cell differentiation into basal stem cells is based on P63 and CK5 expression, goblet cells are based on MUC5AC expression, Clara cells are based on CCSP expression, and ciliated cells are based on FOXJ1 expression It was.

Exemplary differentiation medium
Stage I: Differentiation medium based on definitive endoderm from human ES cells and iPS cells Exemplary RPMI-1640 medium is 2% B-27 (without retinoic acid), 0.1% Albumax II, 1 × Glutamax, 1 × It may contain non-essential amino acids (NEAA), 5 μM LY294002 (PI3K inhibitor), and 100 ng / ml activin A. In some embodiments, 5 μM LY294002 and / or 100 ng / ml activin A is used. However, LY294002 can be used in the range of 2 to 10 μM, for example, and activin A can be used in the range of 75 to 150 ng / ml, for example. In some embodiments, the length of the Stage I treatment period is 4-5 days.

Stage II: Differentiation medium based on RPMI-1640 medium for definitive endoderm to anterior foregut endoderm stage II is 2% B-27 (without retinoic acid), 0.1% Albumax II, 1 × Glutamax, 1 × Non-essential amino acids (NEAA), 0.5-2 μM TGFβ antagonist A8301, 100-500 nM WNT antagonist IWR-1. In some embodiments, the length of the treatment period is 2-4 days.

Stage III: An exemplary RPMI-1640-based differentiation medium for anterior foregut cells to Nkx2.1 + cells stage III is 2% B-27 (with RA), 0.1% Albumax II, 1 × Glutamax, 1 × Non-essential amino acids (NEAA), 20-200 nM BMP4, 20-200 ng / ml FGF2, 5-50 nM GSK3iXV (or 100-1000 nM CHIR-99021 instead of GSK3iXV). In some embodiments, the length of the treatment period is about 4-6 days.

Stage IV: Nkx2.1 early lung endoderm cells to Nkx2.1 + Sox2 + proximal airway and Nkx2.1 + p63 + airway basal stem cells Exemplary RPMI-1640 based media is 2% B-27 (with RA), 0.1% Albumax II, 1 × Glutamax, 1 × Non-essential amino acids (NEAA), 20-100 ng / ml BMP7, 20-100 ng / ml FGF7, 50-100 ng / ml IWR-1 (WNT antagonist), and 1 ˜2 μM PD98059 (MAPKK / ERK antagonist) may be included. In some embodiments, the length of the treatment period is about 4-6 days.

  A second exemplary differentiation medium is shown herein in FIG.

References cited in the examples

Claims (119)

  Isolated human Nkx2.1 positive Sox2 positive proximal airway multipotent progenitor cells.   2. The isolated cell according to claim 1, which is Tuj1 negative and Pax8 negative.   2. The isolated cell of claim 1, which is proliferative and differentiates into airway basal stem cells, ciliated cells, Clara cells, neuroendocrine cells, or squamous epithelial cells under selected differentiation conditions.   Isolated human Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells.   5. The isolated cell according to claim 4, which is FoxP2 positive and / or ID2 positive.   5. The isolated cell of claim 4, which is proliferative and differentiates into any epithelial lung cell when placed in selected differentiation conditions.   Airway basal stem cells, ciliated cells, Clara cells, mucin-secreting goblet cells, type I alveolar cells, type II alveolar cells, squamous epithelial cells, bronchoalveolar stem cells, bronchoalveolar ducts when placed in selected differentiation conditions 7. The isolated cell of claim 6, which differentiates into a junctional stem cell, a migratory CK14 + cell, or a neuroendocrine cell.   Isolated human Nkx2.1 positive p63 positive multipotent airway basal stem cells.   9. The isolated cell of claim 8, which is proliferative and differentiates into ciliated cells, Clara cells, mucin-secreting goblet cells, or basal cells when placed in selected differentiation conditions.   9. The isolated cell of claim 8, wherein the multipotent airway basal stem cell does not express a Clara cell marker, ciliary cell marker, neuroendocrine cell marker, or squamous cell marker.   9. The isolated cell according to claim 8, which is Sox2-positive.   9. The isolated cell according to claim 8, which is CK5 positive and / or NGFR positive.   9. The isolated cell according to claim 1, 5, or 8, which is a disease specific cell.   A composition comprising isolated human Nkx2.1-positive Sox2-positive proximal airway multipotent progenitor cells and a scaffold.   15. The composition of claim 14, wherein the scaffold is implantable in the subject.   15. The composition of claim 14, wherein the cell is autologous to the subject into which the composition is to be implanted.   15. A composition according to claim 14, wherein the scaffold is biodegradable.   15. The composition of claim 14, wherein the scaffold comprises natural fibers, synthetic fibers, decellularized lung tissue, or combinations thereof.   19. The composition of claim 18, wherein the natural fiber is selected from the group consisting of collagen, fibrin, silk, thrombin, chitosan, chitin, alginic acid, hyaluronic acid, and gelatin.   Synthetic fibers are polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactide-co-glycolide) (PLGA), poly (caprolactone), diol / diacid type aliphatic polyester, polyester-amide / Polyester-urethane, poly (valerolactone), poly (hydroxylbutyric acid), polybutylene terephthalate (PBT), polyhydroxyhexanoic acid (PHH), polybutylene succinic acid (PBS), and poly (hydroxyl valeric acid), 20. The composition of claim 19, wherein the composition is selected from the group consisting of representative biodegradable aliphatic polyesters.   15. The composition of claim 14, wherein the proximal airway multipotent progenitor cells are Tuj1 negative and / or Pax8 negative.   A composition comprising isolated human Nkx2.1-positive Sox9-positive distal multipotent lung progenitor cells and a scaffold.   23. The composition of claim 22, wherein the multipotent lung progenitor cells are FoxP2 positive and / or ID2 positive.   24. The composition of claim 22, wherein the scaffold is implantable in the subject.   23. The composition of claim 22, wherein the cell is autologous to the subject into which the composition is to be implanted.   23. The composition of claim 22, wherein the scaffold is biodegradable.   24. The composition of claim 22, wherein the scaffold comprises natural fibers, synthetic fibers, decellularized lungs, or combinations thereof.   28. The composition of claim 27, wherein the natural fiber is selected from the group consisting of collagen, fibrin, silk, thrombin, chitosan, chitin, alginic acid, hyaluronic acid, and gelatin.   Synthetic fibers are polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactide-co-glycolide) (PLGA), poly (caprolactone), diol / diacid type aliphatic polyester, polyester-amide / Polyester-urethane, poly (valerolactone), poly (hydroxylbutyric acid), polybutylene terephthalate (PBT), polyhydroxyhexanoic acid (PHH), polybutylene succinic acid (PBS), and poly (hydroxyl valeric acid), 29. The composition of claim 28, selected from the group consisting of representative biodegradable aliphatic polyesters.   A composition comprising isolated human Nkx2.1-positive p63-positive multipotent airway basal stem cells and a scaffold.   31. The composition of claim 30, wherein the airway basal stem cells are CK5 positive and / or NGFR positive.   32. The composition of claim 30, wherein the scaffold is implantable in the subject.   40. The composition of claim 32, wherein the cell is autologous to the subject into which the composition is to be implanted.   32. The composition of claim 30, wherein the scaffold is biodegradable.   32. The composition of claim 30, wherein the scaffold comprises natural fibers, synthetic fibers, decellularized lung tissue, or combinations thereof.   36. The composition of claim 35, wherein the natural fiber is selected from the group consisting of collagen, fibrin, silk, thrombin, chitosan, chitin, alginic acid, hyaluronic acid, and gelatin.   Synthetic fibers are polylactic acid (PLA), polyglycolic acid (PGA), poly (D, L-lactide-co-glycolide) (PLGA), poly (caprolactone), diol / diacid type aliphatic polyester, polyester-amide / Polyester-urethane, poly (valerolactone), poly (hydroxylbutyric acid), polybutylene terephthalate (PBT), polyhydroxyhexanoic acid (PHH), polybutylene succinic acid (PBS), and poly (hydroxyl valeric acid), 37. The composition of claim 36, selected from the group consisting of representative biodegradable aliphatic polyesters.   31. The composition of claim 14, 22, or 30, wherein the cell is a disease specific cell.   Human foregut endoderm cells are contacted with FGF2, WNT, and BMP4 for a period and at a concentration sufficient to differentiate human foregut endoderm cells into Nkx2.1-positive Tuj1-negative Pax8-negative lung progenitors, respectively. A method of producing a human lung progenitor cell or human lung progenitor cell population that is Nkx2.1 positive, Tuj1 negative, and Pax8 negative, comprising a step.   40. The method of claim 39, wherein the contacting is performed for at least 2 days.   Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells, BMP7, FGF7, WNT antagonist, and MAPKK / ERK antagonist, Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells each Nkx2.1 positive Sox2 positive proximal airway 40. The method of claim 39, further comprising contacting at a sufficient time and concentration to differentiate into multipotent progenitor cells.   42. The method of claim 41, wherein the Wnt antagonist comprises IWR-1.   42. The method of claim 41, wherein the MAPKK / ERK antagonist comprises PD98059.   42. The method of claim 41, wherein the contacting is performed for at least 4 days.   Bk7, FGF7, WNT antagonist, and MAPKK / ERK antagonist are added to Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells, Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells are each Nkx2.1 positive Sox9 positive distal complex 42. The method of claim 41, further comprising contacting at a sufficient time and concentration to differentiate into competent lung progenitor cells.   46. The method of claim 45, wherein the Wnt antagonist comprises IWR-1.   46. The method of claim 45, wherein the MAPKK / ERK antagonist comprises PD98059.   46. The method of claim 45, wherein the contacting is performed for at least 4 days.   40. The method of claim 39, wherein the culture of foregut endoderm cells is derived from embryonic stem (ES) cells or induced pluripotent stem cells (iPSC).   Nkx2.1-positive Tuj1-negative Pax8-negative cells cultured with B27, BMP7, FGF7, and WNT antagonist, Nkx2.1-positive Tuj1-negative Pax8-negative lung progenitor cells, Nkx2.1-positive p63-positive multipotent airway basal stem cells 40. The method of claim 39, further comprising contacting at a sufficient time and concentration to differentiate into.   40. The method of claim 39, further comprising contacting Noggin with a culture of Nkx2.1 positive Tuj1 negative Pax8 negative cells.   51. The method of claim 50, wherein the contacting is performed for at least 10 days.   A method comprising administering a composition comprising an isolated human lung progenitor cell and a pharmaceutically acceptable carrier to a subject having a lung disease or disorder or injury, comprising: How to treat.   Isolated human lung progenitor cells are Nkx2.1 positive Sox2 positive proximal airway multipotent progenitor cells, Nkx2.1 positive Sox9 positive distal multipotent progenitor cells, Nkx2.1 positive p63 positive multipotent airway basal stem cells 54. The method of claim 53, wherein the method is selected from the group consisting of:   54. The method of claim 53, wherein the proximal airway multipotent progenitor cells are Tuj1 negative and / or Pax8 negative.   54. The method of claim 53, wherein the distal multipotent lung progenitor cells are FoxP2 and / or ID2 positive.   54. The method of claim 53, wherein the airway basal stem cells are CK5 positive and / or NGFR positive.   54. The method of claim 53, wherein the composition is administered to the lung.   54. The method of claim 53, wherein the isolated human lung progenitor cells are autologous to the subject to which the composition is administered.   54. The method of claim 53, wherein the composition further comprises a scaffold.   64. The method of claim 60, wherein the scaffold is implantable in the subject.   64. The method of claim 60, wherein the scaffold is biodegradable.   61. The method of claim 60, wherein the scaffold comprises natural fibers, synthetic fibers, decellularized lung tissue, or combinations thereof.   64. The method of claim 60, wherein the scaffold comprises a substance that promotes differentiation of isolated human lung progenitor cells.   64. The method of claim 60, wherein the composition is formulated for aerosol delivery. (A) culturing a human disease-specific airway cell population produced by in vitro differentiation of human disease-specific lung progenitor cells in the presence and absence of a candidate substance for treating a lung disease or disorder,
(B) comparing the expression or activity of at least one marker up-regulated in the disease in the presence and absence of the candidate substance, or the expression or activity of at least one marker down-regulated in the disease Lungs in a subject if the expression or activity of at least one up-regulated disease marker decreases or the expression or activity of at least one down-regulated disease marker increases A method for screening a substance for treating a pulmonary disease or disorder comprising the step of identifying a candidate substance as a candidate for treating a disease or pulmonary disorder.   68. The method of claim 66, further comprising the step of differentiating the isolated human disease specific lung progenitor cell population into a culture of human disease specific airway cells prior to step (a).   66. Prior to step (a), further comprising the step of differentiating an induced pluripotent stem cell population derived from a subject having a lung disease or disorder into an isolated human disease-specific lung progenitor cell population. The method described in 1.   68. The method of claim 66, wherein the candidate substance comprises a small molecule, protein, polypeptide, antibody or antigen-binding fragment thereof, or nucleic acid.   Human lung progenitor cells are human Nkx2.1 positive Sox2 positive proximal airway multipotent progenitor cells, human Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells, and human Nkx2.1 positive p63 positive multipotent airways 68. The method of claim 66, wherein the method is selected from the group consisting of basal stem cells.   Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells, BMP7, FGF7, WNT antagonist, and MAPKK / ERK antagonist, Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells each Nkx2.1 positive Sox9 positive proximal airway Human Nkx2.1 positive Sox2 positive proximal airway by a method comprising contacting at a sufficient time and concentration to differentiate into multipotent progenitor cells or Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells 72. The method of claim 70, wherein multipotent progenitor cells and human Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells are generated.   72. The method of claim 71, wherein the contacting is performed for at least 4 days.   Nkx2.1 positive Tuj1 negative Pax8 negative cells, B27, BMP7, FGF7, and WNT antagonist, each of which converts Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells to Nkx2.1 positive p63 positive multipotent airway basal stem cells 71. The method of claim 70, wherein human Nkx2.1 positive p63 positive multipotent airway basal stem cells are produced by a method comprising contacting at a sufficient time and concentration for differentiation.   74. The method of claim 73, further comprising contacting Noggin with Nkx2.1 positive Tuj1 negative Pax8 negative cells.   74. The method of claim 73, wherein the contacting is performed for at least 10 days. (A) culturing Nkx2.1-positive Tuj1-negative Pax8-negative human lung progenitor cells in the presence and absence of candidate differentiation substances,
(B) comparing the expression or activity of at least one marker that is up-regulated upon differentiation of lung progenitor cells to a more differentiated state in the presence and absence of candidate substances, or lung progenitor cells Comparing the expression or activity of at least one marker that is down-regulated upon differentiation to a more differentiated state, wherein the expression or activity of at least one up-regulated differentiation marker decreases Or an increase in the expression or activity of at least one down-regulated differentiation marker indicates that the candidate substance can be used to induce differentiation of isolated human lung progenitor cells in the subject. A method for screening a substance that induces differentiation of human lung progenitor cells.   77. The method of claim 76, further comprising differentiating embryonic stem cells or induced pluripotent stem cells into human lung progenitor cells prior to step (a).   77. The method of claim 76, wherein the candidate substance comprises a small molecule, protein, polypeptide, antibody, or nucleic acid.   Human lung progenitor cells are human Nkx2.1 positive Sox2 positive proximal airway multipotent progenitor cells, human Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells, and human Nkx2.1 positive p63 positive multipotent airways 77. The method of claim 76, selected from the group consisting of basal stem cells.   Nkx2.1 positive Tuj1 negative Pax8 negative human lung progenitor cells, BMP7, FGF7, WNT antagonist, and MAPKK / ERK antagonist, Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells, Nkx2.1 positive Sox9 positive proximal Human Nkx2.1 positive Sox2 positive proximal by a method comprising contacting with sufficient duration and concentration to differentiate into airway multipotent progenitor cells or Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells 80. The method of claim 79, wherein airway multipotent progenitor cells and human Nkx2.1 positive Sox9 positive distal multipotent lung progenitor cells are generated.   81. The method of claim 80, wherein the contacting is performed for at least 4 days.   Nkx2.1 positive Tuj1 negative Pax8 negative cells, B27, BMP7, FGF7, and WNT antagonist, each of which converts Nkx2.1 positive Tuj1 negative Pax8 negative lung progenitor cells to Nkx2.1 positive p63 positive multipotent airway basal stem cells 80. The method of claim 79, wherein human Nkx2.1 positive p63 positive multipotent airway basal stem cells are generated by a method comprising contacting at a sufficient time and concentration for differentiation.   84. The method of claim 82, further comprising contacting Noggin with Nkx2.1 positive Tuj1 negative Pax8 negative cells.   84. The method of claim 82, wherein the contacting is performed for at least 10 days.   A kit for treating a pulmonary disease or disorder comprising the cell of claim 1, 5 or 9, a pharmaceutically acceptable carrier, and instructions for treating the pulmonary disease or disorder.   86. The kit of claim 85, further comprising a scaffold.   A kit for screening a candidate substance, comprising the cell according to claim 1, 5, or 8, one or more substances for detecting a lung-specific cell surface marker, and instructions thereof.   90. The kit of claim 87, further comprising a cell culture medium, a growth factor, and / or a differentiation substance. (I) two or more of BMP4, FGF2, WNT, BMP7, FGF7, WNT antagonist, PI3 kinase inhibitor, activin A, Noggin, B27, and retinoic acid, optionally provided in unit dose;
(Ii) optionally a cell culture medium;
(Iii) one or more substances for detecting lung cell specific surface markers; and (iii) a kit for differentiating human stem cells into human lung progenitor cells, comprising instructions thereof.   A cell or tissue medium comprising B27, activin A, and ZSTK474.   The medium according to claim 90, wherein the concentration of B27 is 1% to 5%.   92. The medium of claim 91, wherein the concentration of B27 is 2%.   The medium according to claim 90, wherein the concentration of activin A is 10 to 40 ng / mL.   94. The medium of claim 93, wherein the activin A concentration is 20 ng / mL.   The medium according to claim 90, wherein the concentration of ZSTK474 is 0.2 to 0.5 µM.   92. The medium of claim 90, comprising a component of RPMI medium.   A cell or tissue medium comprising CHIR9902, PIK-75, dorsomorphin, and FGF2.   98. The medium of claim 97, wherein the concentration of CHIR9902 is in the range of 0.1-1 μM.   98. The medium of claim 97, wherein the concentration of PIK-75 comprises 0.01-0.1 [mu] M.   98. The medium of claim 97, wherein the concentration of dorsomorphin comprises 1-5 [mu] M.   98. The medium of claim 97, wherein the concentration of FGF2 comprises 10-100 ng / mL.   Further comprising a drug selected from the group consisting of GF-109203X, Ro31-8220, Pp242, PIK-75, ZSTK474, PMA, carvedilol, corticosterone, triclabendazole, benproperin phosphate, phenothiazine, and methotrexate. 97. The medium according to 97.   In human foregut endoderm cells, Wnt agonist, PIK3 kinase inhibitor, BMP antagonist, GF-109203X, Ro31-8220, Pp242, PIK-75, ZSTK474, PMA, carvedilol, corticosterone, triclabendazole, phosphate A drug selected from the group consisting of benproperin, phenothiazine, and methotrexate is contacted for a period and at a concentration sufficient for each to differentiate human foregut endoderm cells into Nkx2.1-positive Tuj1-negative Pax8-negative lung progenitor cells A method of producing a human lung progenitor cell or human lung progenitor cell population that is Nkx2.1 positive, Tuj1 negative, and Pax8 negative, comprising a step.   105. The method of claim 104, wherein the contacting is performed for at least 2 days.   105. The method of claim 104, wherein the Wnt agonist comprises CHIR9902.   107. The method of claim 106, wherein the concentration of CHIR9902 is in the range of 0.1-1 μM.   105. The method of claim 104, wherein the PI3 kinase inhibitor comprises PIK-75.   109. The method of claim 108, wherein the concentration of PIK-75 comprises 0.01-0.1 [mu] M.   105. The method of claim 104, wherein the BMP antagonist comprises dorsomorphin.   111. The method of claim 110, wherein the concentration of dorsomorphin comprises 1-5 [mu] M.   105. The method of claim 104, wherein the growth factor comprises FGF2.   113. The method of claim 112, wherein the concentration of FGF2 comprises 10-100 ng / mL.   A definitive endoderm cell or definitive endoderm comprising contacting BPS, activin A, and ZSTK474 with iPSC or ESC for a period and at a concentration sufficient to each differentiate iPSC or ESC into definitive endoderm cells. A method of producing a cell population.   115. The method of claim 114, wherein production of definitive endoderm cells is determined by FOXA2 / SOX17 co-staining or by FACS analysis with a combination of cKit / CXCR4 and / or cKit / EpCAM.   115. The method of claim 114, wherein the concentration of B27 is 1% to 5%.   117. The method of claim 116, wherein the concentration of B27 is 2%.   115. The method of claim 114, wherein the concentration of activin A is 10-40 ng / mL.   119. The method of claim 118, wherein the activin A concentration is 20 ng / mL.   115. The method of claim 114, wherein the concentration of ZSTK474 is 0.2-0.5 [mu] M.

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